Synovial extracellular matrix-specific chimeric antigen receptor for targeting regulatory t cells to treat autoimmune diseases

ABSTRACT

Disclosed herein are chimeric antigen receptors (“CARs”) comprising an antigen binding site that recognizes citrullinated polypeptides. Citrullinated polypeptides, such as citrullinated vimentin, fibrinogen, and filaggrin, are expressed in the synovium of subjects with rheumatoid arthritis. Further disclosed are T cells, and in particular, Treg cells, that express these CARs. Administration of these CAR-T cells is useful in the treatment of rheumatoid arthritis as well as other diseases associated with citrullinated peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/227,320, filed Jul. 29, 2021, and U.S. Provisional Application No.63/339,361, filed May 6, 2022, each of which is hereby incorporated byreference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(237752000340SEQLIST.xml; Size: 50,843 bytes; and Date of Creation: Jul.28, 2022) is herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to chimeric antigen receptors reactivewith citrullinated antigens and regulatory T cells expressing thereceptors for treating autoimmune disease.

BACKGROUND

Autoimmune diseases affect a significant number of people. For example.Rheumatoid Arthritis (RA) is a chronic inflammatory disease targetingperipheral joints leading to bone erosion, impairment of mobility, anddecreased quality of life. It is affecting 0.5-1% of the populationworldwide and the incidence rate keeps rising. The pathogenesis of RA ismainly localized in the synovial joint where immune cells composed of Tcells, B cells, macrophages, and dendritic cells infiltrate thesynovium. Moreover, fibroblast-like synoviocytes present in thesublining layer of the synovium proliferate and contribute to cartilagedamage.

Synovial hyperplasia in rheumatoid arthritis results in infiltration ofthe synovium by immune cells, and subsequent cartilage damage and boneerosion.

Currently there is no cure for RA as well as for many other autoimmuneconditions. Lifelong treatment is usually required for patients with RA,which in addition of being extremely expensive, may cause severe sideeffects in the long term such as infections and rheumatoid arthritisrisk.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate exemplary embodiments and, togetherwith the description, further serve to enable a person skilled in thepertinent art to make and use these embodiments and others that will beapparent to those skilled in the art. The invention will be moreparticularly described in conjunction with the following drawingswherein:

FIG. 1 : Initial data comprising an MND promoter and EGFRt backboneshowed that CV CARs BVCA1 and SBT01G had strongest response toplate-bound full-length CV (n=2).

FIG. 2 : Assay with soluble full-length CV showed a dose response onlyfor CV CARs BVCA1 and SBT01G-HL (n=1).

FIG. 3 : Assay with soluble bead-bound-peptide demonstrated bindingspecificity (n=1).

FIG. 4 : MND-SBT01G shows a stronger response to plate-bound, antibodycaptured CV, but not soluble CV than MND-BVCA1.

FIG. 5 : Testing of an initial batch of synovial fluid from InnovativeResearch showed SBT01G gave stronger responses than BVCA1.

FIG. 6 : Further testing of 15 synovial fluid samples from Swedishpatients showed that, for samples that gave a response, SBT01G wasstronger than BVCA1.

FIG. 7 : Primary Treg responses to synovial fluid (SF) demonstrateSBT01G is more sensitive than BVCA1 to SF from RA patients.

FIG. 8 : SBT01G, but not BVCA1, is also able to respond to plate-boundfull-length PAD2 citrullinated fibrinogen.

FIG. 9 SBT01G CAR as effectors and Tregs respond to CitrullinatedFibrinogen but BVCA1 does not.

FIG. 10A-10B: Both EF1A and MND promoters demonstrate functionalresponses by CV CARs to soluble bead-bound-peptide. Thus, the CARpromoter does not influence Treg phenotype.

FIG. 11 : scFv linker has little to no impact on SBT01G CAR-basedfunction.

FIG. 12A-12B: SBT01G is expressed by a higher percentage of cells thanBVCA1, but BVCA1 and SBT01G CAR-T cells have similar FoxP3 and Heliosprofiles.

FIG. 13 : CV-CAR Treg cells (SBT01G) but not untransduced Treg cells areactivated by citrullinated vimentin (CV). Activation was demonstrated bytarget antigen-specific increases in proliferation, CD71 expression andIL-10 secretion.

FIG. 14 : CV-CAR Treg cells respond to citrullinated proteins in thesynovial fluid from the majority of RA patients. In contrast, CV-CARTreg cells do not respond to synovial fluid from normal controls(subjects without RA).

FIG. 15 : CV-CAR Treg cells (SBT01G) but not untransduced Treg cellsfrom two donors are specifically activated by synovial fluid from RApatients.

FIG. 16A-16B: Assessment of the suppressive function of CV-CAR Tregcells. FIG. 16A shows that CV-CAR Treg cells are able to suppressproliferation of CD3/CD28-preactivated Teff cells in the presence butnot the absence of CV. FIG. 16B shows that CV-CAR Treg cells are able tosuppress proliferation of CD19-CAR Teff cells in the presence of CV,whereas untransduced Treg cells are not.

FIG. 17 : A timeline of human CV-CAR Treg cell activation in vivo in alipopolysaccharide (LPS)-induced, murine model of pulmonary inflammationis shown. In brief, human CV-CAR Treg cells were administeredintravenously (IV) on Day 0, human IL-2 was administeredintraperitoneally (IP) twice daily, and LPS was administeredintranasally (IN) on Days 0, 1, 6 and 12. On Day 13, mice weresacrificed and organs were harvested to facilitate analysis of Tregcells.

FIG. 18A-18B: Flow cytometry dot plots comparing epidermal growth factor(EGFR) expression by human CV-CAR Treg cells versus of levels of CellTrace Violet (CTV) are shown. FIG. 18A shows how a proliferation ratioof the CV-CAR Tregs (EGFR+) was determined. Specifically, theproliferation ratio equals the % of EGFR+, CTV− cells divided by the %of EGFR+, CTV− cells. FIG. 18B shows how a fold change in EGFR ratio wasdetermined.

FIG. 19A-19B: CV-CAR Tregs proliferate in a LPS-induced, murine model ofpulmonary inflammation but not in control recipients of PBS. FIG. 19Ashows the absolute number of CD45+, CD3+ Tregs in the lungs of variousstudy groups, while FIG. 19B shows the proliferation ratio of the Tregsin the lungs of various study groups.

SUMMARY

Regulatory T cells (Tregs) are defective in patients and mouse models ofRA. Therefore, Treg-based adoptive cell therapy (ACT) represents apromising approach in RA. In fact, Treg-based ACT reverses disease inanimal models of RA. In this study, an antibody isolated from a RApatient was used to engineer a CAR specific for citrullinated vimentin(CV) and other posttranslational modified proteins found abundantly andalmost exclusively in the synovial extracellular matrix (ECM) ofaffected joints.

Disclosed herein are chimeric antigen receptors (CAR) which specificallyrecognize antigens associated with autoimmune diseases. In particular,the CARs can be specific for post-translationally modified antigens. Inparticular, the CARs can be specific for binding to citrullinatedpolypeptides, including vimentin, citrullinated filaggrin andcitrullinated fibrinogen.

Chimeric antigen receptors (CAR) were engineered to specifically targetpost-translationally modified proteins, namely citrullinated vimentin,citrullinated filaggrin and citrullinated fibrinogen, that are expressedin the extracellular matrix of inflamed joints in patients withRheumatoid Arthritis (RA). In some embodiments, the single chainfragment variable (scFv) part of the CAR is obtained from an antibodyhighly specific for citrullinated proteins isolated from the peripheralblood of an RA patient. In one embodiment, specific scFv chains wereinserted into a second-generation CAR construct. In some embodiments,the scFv chains were inserted into a CAR construct cloned in alentiviral vector. In the detailed description, references to antibodiesare applicable to antigen-binding domains of the CARs of the presentdisclosure unless the context indicates otherwise.

DETAILED DESCRIPTION I. Definitions

Unless otherwise specified, terms and symbols of biochemistry, nucleicacid chemistry, molecular biology, developmental biology and moleculargenetics follow those of standard treaties and texts in the field, forexample, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2ndEdition (Cold Spring Harbor Press, 1989); Alberts and Singer,Developmental Biology, Eighth Edition (Sinauer Associates Inc.,Sunderland, MA, 2006); Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Gaits, ed., OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Lehninger,Biochemistry, Second Edition (Worth Publishers, New York, 1975);Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); and the like.

As used herein, the terms “antigen,” “immunogen,” and “antibody target,”refer to a molecule, compound, or complex that is recognized by anantibody, i.e., can be bound by the antibody. The term can refer to anymolecule that can be recognized by an antibody, e.g., a polypeptide,polynucleotide, carbohydrate, lipid, chemical moiety, or combinationsthereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Oneof skill will understand that the term does not indicate that themolecule is immunogenic in every context, but simply indicates that itcan be targeted by an antibody.

As used herein, the term “epitope” refers to the localized site on anantigen that is recognized and bound by an antibody. Epitopes caninclude a few amino acids or portions of a few amino acids, e.g., 5 or6, or more, e.g., 20 or more amino acids, or portions of those aminoacids. In some cases, the epitope includes non-protein components, e.g.,from a carbohydrate, nucleic acid, or lipid. In some cases, the epitopeis a three-dimensional moiety. Thus, for example, where the target is aprotein, the epitope can be comprised of consecutive amino acids, oramino acids from different parts of the protein that are brought intoproximity by protein folding (e.g., a discontinuous epitope).

As used herein, the term “antibody” refers to a polypeptide comprising aframework region from an immunoglobulin gene, that specifically bind andrecognize an antigen. Typically, the “variable region” contains theantigen-binding region of the antibody (or its functional equivalent)and is most critical in specificity and affinity of binding. Anexemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD).

Antibodies can be of (i) any of the five major classes ofimmunoglobulins, based on the identity of their heavy-chain constantdomains—alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu(IgM), or (ii) subclasses (isotypes) thereof (E.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2). The light chains can be either lambda or kappa.

The following are a non-exhaustive list of different antibody forms, allretaining antigen binding activity:

-   -   (1) whole immunoglobulins (also referred to as “intact”        antibodies) (two light chains and two heavy chains, e.g., a        tetramer);    -   (2) an immunoglobulin polypeptide (a light chain or a heavy        chain);    -   (3) an antibody fragment, such as Fv (a monovalent or bi-valent        variable region fragment, and can encompass only the variable        regions (e.g., V_(L) and/or V_(H)), Fab (V_(L)C_(L) V_(H)C_(H)),        F(ab′)2, Fv (V_(L)V_(H)), scFv (single chain Fv) (a polypeptide        comprising a V_(L) and V_(H) joined by a linker, e.g., a peptide        linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific        (scFv)2, minibody (sc(FV)2 fused to CH3 domain), diabody        (noncovalent dimer of single-chain Fv (scFv) fragment that        consists of the heavy chain variable (VH) and light chain        variable (VL) regions connected by a small peptide linker),        triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3;    -   (4) a multivalent antibody (an antibody comprising binding        regions that bind two different epitopes or proteins, e.g.,        “scorpion” antibody;    -   (5) a fusion protein comprising a binding portion of an        immunoglobulin fused to another amino acid sequence (such as a        fluorescent protein); and    -   (6) heavy chain only antibody or antibody fragment having only        two heavy chains and lacking the two light chains usually found        in antibodies.

Production and properties of tandem scFvs and diabodies are described,e.g., in Asano et al. (2011) J Biol. Chem. 286:1812; Kenanova et al.(2010) Prot Eng Design Sel 23:789; Asano et al. (2008) Prot Eng DesignSel 21:597.

The phrase “CDR sequence set” as used herein refers to the 3 heavy chainand/or 3 light chain CDRs of a particular antibody described herein. A“light chain” CDR sequence set refers to the light chain CDR sequences.A “heavy chain” CDR sequence set refers to the heavy chain CDRsequences. A “full” CDR sequence set refers to both heavy chain andlight chain CDR sequences. CDRs are predicted based on IMGT sequencealignment.

As used herein, the term “chimeric antibody” refers to an antibodyhaving amino acid sequences derived from two or more species. In oneembodiment, the variable region of both light and heavy chainscorrespond to the variable region of antibodies derived from one speciesof mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity,affinity and capability, while the constant region are homologous thesequence derived from another species (typically in the subjectreceiving the therapy, e.g., human) to avoid eliciting an immuneresponse.

As used herein, the term “humanized antibody” refers to a chimericantibody in which the CDRs, obtained from the VH and VL regions of anon-human antibody having the desired specificity, affinity andcapability are grafted to a human framework sequence. In one embodiment,the framework residues of the humanized antibody are modified to refineand optimize the antibody specificity, affinity and capability.Humanization, i.e., substitution of non-human CDR sequences for thecorresponding sequences of a human antibody, can be performed followingthe methods described in, e.g., U.S. Pat. Nos. 5,545,806; 5,569,825;5,633,425; 5,661,016; Riechmann et al., Nature 332:323-327 (1988); Markset al., Bio/Technology 10:779-783 (1992); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996).

As used herein, the term “human antibody” refers to an antibody producedby a human or an antibody having an amino acid sequence correspondingthereto made by any technique known in the art.

The specificity of the binding can be defined in terms of thecomparative dissociation constants (Kd) of the antibody (or othertargeting moiety) for target, as compared to the dissociation constantwith respect to the antibody and other materials in the environment orunrelated molecules in general. A larger (higher) Kd is a Kd thatdescribes a lower affinity interaction. Conversely a smaller (lower) Kdis a Kd that describes a higher affinity interaction or tighter binding.By way of example only, the Kd for an antibody specifically binding to atarget may be femtomolar, picomolar, nanomolar, or micromolar and the Kdfor the antibody binding to unrelated material may be millimolar orhigher. Binding affinity can be in the micromolar range (kD=10⁻⁴ to10⁻⁶), nanomole range (kD=10⁻⁷ M to 10⁻⁹ M), picomole range (kD=10⁻¹⁰ Mto 10⁻¹² M), or femtomole range (kD=10⁻¹³ M to 10⁻¹⁵ M).

As used herein, an antibody “binds” or “recognizes” an antigen orepitope if it binds the antigen or epitope with a Kd of less than 10⁻⁴ M(i.e., in the micromolar range). The term “binds” with respect to a celltype (e.g., an antibody that binds cancer cells), typically indicatesthat an agent binds a majority of the cells in a pure population ofthose cells. For example, an antibody that binds a given cell typetypically binds to at least ⅔ of the cells in a population of theindicated cells (e.g., 67, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100%). In some cases, binding to a polypeptide can be assayedby comparing binding of the antibody to a cell that presents thepolypeptide to binding (or lack thereof) of the antibody to a cell thatdoes not express the polypeptide. One of skill will recognize that somevariability will arise depending on the method and/or threshold ofdetermining binding. Affinity of an antibody for a target can bedetermined according to methods known in the art, e.g., as reviewed inErnst et al. Determination of Equilibrium Dissociation Constants,Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

As used herein, the term “greater affinity” as used herein refers to arelative degree of antibody binding where an antibody X binds to targetY more strongly (Kon) and/or with a smaller dissociation constant (Koff)than to target Z, and in this context antibody X has a greater affinityfor target Y than for Z. Likewise, the term “lesser affinity” hereinrefers to a degree of antibody binding where an antibody X binds totarget Y less strongly and/or with a larger dissociation constant thanto target Z, and in this context antibody X has a lesser affinity fortarget Y than for Z. The affinity of binding between an antibody and itstarget antigen, can be expressed as KA equal to 1/KD where KD is equalto kon/koff. The kon and koff values can be measured using surfaceplasmon resonance technology, for example, using a Molecular AffinityScreening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). Anantagonist or blocking antibody is an antibody that partially or fullyblocks inhibits or neutralizes a biological activity related to thetarget antigen relative to the activity under similar physiologicalconditions when the antibody is not present. Antagonists can becompetitive, non-competitive or irreversible. A competitive antagonistis a substance that binds to a natural ligand or receptor at the samesite as the natural ligand-receptor interaction or binds allostericallyin a manner that induces a change to prevent normal binding. Anon-competitive antagonist binds at a different site than the naturalligand-receptor interaction, but lower the KD or signal resulting fromthe interaction. An irreversible inhibitor causes covalent modificationsto the receptor preventing any subsequent binding.

As used herein, the term “avidity” refers to the overall stability ofthe binding complex between the antibody and the target antigen. It isgoverned by three factors, (i) the intrinsic affinity of the antibodyfor the antigen, (2) the valency of the antibody, and (3) the geometricarrangement of the interacting components. Affinity is the strength ofthe interaction between the antibody and a single target, whereasavidity is an accumulated strength of multiple affinities. In oneembodiment, the antibodies provided herein are divalent.

As used herein, an antibody “preferentially binds” binds a first antigenrelative to a second antigen if it binds the first antigen with greateraffinity than it does the second antigen. Preferential binding can be atleast any of 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 100-fold, 500-fold or 1000-fold greater affinity.

As used herein, an antibody “specifically binds” or is “specific for” atarget antigen or target group of antigens if it binds the targetantigen or each member of the target group of antigens with an affinityof at least any of 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M,1×10⁻¹¹ M, 1×10⁻¹² M, and, for example, binds to the target antigen oreach member of the target group of antigens with an affinity that is atleast two-fold greater than its affinity for non-target antigens towhich it is being compared. Typically, specific binding is characterizedby binding the antigen with sufficient affinity that the antibody isuseful as a diagnostic to detect the antigen or epitope and/or as atherapeutic agent in targeting the antigen or epitope.

As used herein, the term “polypeptide” refers to a molecule having asequence of natural and/or unnatural amino acids connected throughpeptide bonds. The term “peptide” refers to a short polypeptide,typically no more than 30 amino acids long. The amino acid sequence of apolypeptide is referred to as its “primary structure.” The term“protein” refers to a polypeptide having a secondary, tertiary and/orquaternary structure, e.g., structures stabilized by hydrogen bonds,relationships between secondary structures and structures formed of morethan one protein. Proteins can be further modified by other attachedmoieties such as carbohydrate (glycoproteins), lipids (lipoproteins)phosphate groups (phosphoproteins) and the like.

As used herein, an amino acid sequence “consists of” only the aminoacids in that sequence.

As used herein, a first amino acid sequence “consists essentially of” asecond amino acid sequence if the first amino acid sequence (1)comprises the second amino sequence and (2) is no more than 1, no morethan 2 or no more than 3 amino acids longer than the second amino acidsequence.

As used herein, a first amino acid sequence is a “fragment” of a secondamino acid sequence if the second amino acid sequence comprises thefirst amino acid sequence. In certain embodiments, a first amino acidsequence that is a fragment of a second amino acid sequence may have nomore than any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fewer amino acids thanthe second amino acid sequence.

As used herein, a “functional equivalent” of a reference amino acidsequence is a sequence that is not identical to the reference sequence,but that contains minor alterations such as, for example, insertion,deletion or substitution of one or a few amino acids. A functionallyequivalent sequence retains the function (e.g., immunogenicity) of thereference sequence to which it is equivalent. If a functionallyequivalent amino acid sequence contains substitution of one or moreamino acids with respect to the reference sequence, these will generallybe conservative amino acid substitutions.

As used herein, a “conservative amino acid substitution” is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. See, e.g., Watson, et al., “Molecular Biology of the Gene,” 4thEdition, 1987, The Benjamin/Cummings Pub. Co., Menlo Park, CA, p. 224.Examples of conservative amino acid substitution include the following(Note, some categories are not mutually exclusive):

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Ala, Val, Ile, Leu, Met, Gly, Pro (non-polar, hydrophobic)Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

As used herein, the term “substantially identical” refers to identitybetween a first amino acid sequence that contains a sufficient orminimum number of amino acid residues that are i) identical to, or ii)conservative substitutions of aligned amino acid residues in a secondamino acid sequence such that the first and second amino acid sequenceshave a common structural domain and/or common functional activity and/orcommon immunogenicity. For example, amino acid sequences that contain acommon structural or antigenic domain having at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are termedsufficiently or substantially identical. In the context of nucleotidesequence, the term “substantially identical” is used herein to refer toa first nucleic acid sequence that contains a sufficient or minimumnumber of nucleotides that are identical to aligned nucleotides in asecond nucleic acid sequence such that the first and second nucleotidesequences encode a polypeptide having common functional activity, orencode a common structural polypeptide domain or a common functionalpolypeptide activity, or encode polypeptides having the same immunogenicproperties.

As used herein, a chemical entity, such as a polypeptide, is“substantially pure” or “isolated” if it is the predominant chemicalentity of its kind (e.g., of polypeptides) in a composition. Thisincludes the chemical entity representing more than 50%, more than 80%,more than 90%, more than 95%, more than 98%, more than 99%, more than99.5%, more than 99.9%, or more than 99.99% of the chemical entities ofits kind in the composition. A substantially purified fraction is acomposition wherein the object species comprises at least about 50% (ona molar basis) of all macromolecular species present. Generally, asubstantially pure composition means that about 80% to 90% or more ofthe macromolecular species present in the composition is the purifiedspecies of interest. The object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) if the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules, stabilizers (e.g., BSA), and elemental ion species are notconsidered macromolecular species for purposes of this definition.

The phrase “isolated antibody” refers to antibody produced in vivo or invitro that has been removed from the source that produced the antibody,for example, an animal, hybridoma or other cell line (such asrecombinant insect, yeast or bacterial cells that produce antibody).

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score—50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

For antibodies, percentage sequence identities can be determined whenantibody sequences maximally aligned by IMGT. After alignment, if asubject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,multiplied by 100 to convert to percentage.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program NCBI-BLAST2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. The term “nucleic acid sequence”as used herein refers to a sequence of nucleoside or nucleotide monomersconsisting of naturally occurring bases, sugars and intersugar(backbone) linkages and includes cDNA. The term also includes modifiedor substituted sequences comprising non-naturally occurring monomers orportions thereof. The nucleic acid sequences of the present applicationmay be deoxyribonucleic acid sequences (DNA) or ribonucleic acidsequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. It is understood that polynucleotides comprisingnon-transcribable nucleotide bases may be useful as probes in, forexample, hybridization assays. The nucleic acid can be either doublestranded or single stranded, and represents the sense or antisensestrand. Further, the term “nucleic acid” includes the complementarynucleic acid sequences as well as codon optimized or synonymous codonequivalents.

The term “isolated nucleic acid” as used herein refers to a nucleic acidsubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences that naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10[Na+])+0.41(%(G+C)−600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example, if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5× Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

As used herein, the term “expression construct” refers to apolynucleotide comprising an expression control sequence operativelylinked with a heterologous nucleotide sequence (i.e., a sequence towhich the expression control sequence is not normally connected to innature) that is to be the subject of expression. As used herein, theterm “expression vector” refers to a polynucleotide comprising anexpression construct and sequences sufficient for replication in a hostcell or insertion into a host chromosome. Plasmids and viruses areexamples of expression vectors. As used herein, the term “expressioncontrol sequence” refers to a nucleotide sequence that regulatestranscription and/or translation of a nucleotide sequence operativelylinked thereto. Expression control sequences include promoters,enhancers, repressors (transcription regulatory sequences) and ribosomebinding sites (translation regulatory sequences).

As used herein, a nucleotide sequence is “operatively linked” with anexpression control sequence when the expression control sequencefunctions in a cell to regulate transcription of the nucleotidesequence. This includes promoting transcription of the nucleotidesequence through an interaction between a polymerase and a promoter.

The term “vector” as used herein comprises any intermediary vehicle fora nucleic acid molecule which enables said nucleic acid molecule, forexample, to be introduced into prokaryotic and/or eukaryotic cellsand/or integrated into a genome, and include plasmids, phagemids,bacteriophages or viral vectors such as retroviral based vectors,lentiviral vectors, Adeno Associated viral vectors and the like. Theterm “plasmid” as used herein generally refers to a construct ofextrachromosomal genetic material, usually a circular DNA duplex, whichcan replicate independently of chromosomal DNA.

“Transfection” refers to the introduction of new genetic material into acell. It includes transformation (the direct uptake and incorporation ofexogenous genetic material from its surroundings through the cellmembrane), transduction (the introduction of foreign DNA by abacteriophage virus into a host cell) and conjugation.

As used herein, a “host cell” refers to a recombinant cell comprising anexpression construct.

As used herein, the term “biological sample” refers to a samplecontaining cells (e.g., tumor cells) or biological molecules derivedfrom cells.

As used herein, the term terms “therapy,” “treatment,” “therapeuticintervention” and “amelioration” refer to any activity resulting in areduction in the severity of symptoms. The terms “treat” and “prevent”are not intended to be absolute terms. Treatment and prevention canrefer to any delay in onset, amelioration of symptoms, improvement inpatient survival, increase in survival time or rate, etc. Treatment andprevention can be complete or partial. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment, or to the same patient prior to treatment or at a differenttime during treatment. In some aspects, the severity of disease isreduced by at least 10%, as compared, e.g., to the individual beforeadministration or to a control individual not undergoing treatment. Insome aspects, the severity of disease is reduced by at least 25%, 50%,75%, 80%, or 90%, or in some cases, no longer detectable using standarddiagnostic techniques. “Treating” and “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. “Treating” and “treatment” as used herein also includeprophylactic treatment.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited (e.g.,open-ended terms meaning including but not limited to). For example, acomposition that “comprises” or “includes” an antibody may contain theantibody alone or in combination with other ingredients. In contrast,the phrase “consisting of” is closed, indicating that such embodimentsdo not include additional elements. The term “consisting essentially of”refers to the inclusion of recited elements and other elements that donot materially affect the basic and novel characteristics of a claimedcombination (e.g., partially closed term). It is understood that aspectsand embodiments described herein as “comprising” include “consisting of”and “consisting essentially of” embodiments.

As used herein, the following meanings apply unless otherwise specified.The word “may” is used in a permissive sense (i.e., meaning having thepotential to), rather than the mandatory sense (i.e., meaning must). Thesingular forms “a,” “an,” and “the” include plural referents. Thus, forexample, reference to “an element” includes a combination of two or moreelements, notwithstanding use of other terms and phrases for one or moreelements, such as “one or more.” The phrase “at least one” includes“one”, “one or more”, “one or a plurality” and “a plurality”. The term“or” is, unless indicated otherwise, non-exclusive, i.e., encompassingboth “and” and “or.” The term “any of” between a modifier and a sequencemeans that the modifier modifies each member of the sequence. So, forexample, the phrase “at least any of 1, 2 or 3” means “at least 1, atleast 2 or at least 3”.

II. Chimeric Antigen Receptors

“Chimeric antigen receptors” or “CARs” are engineered moleculescomprising an optional signal peptide, a target binding domain, anoptional hinge region, a transmembrane domain, an intracellularsignaling domain and an optional co-stimulatory domain. CARs are basedon the structure of T cell receptors, which are expressed on T cells andwhich are involved in the cell-mediated immune response. The “targetbinding domain” is also referred to herein as an “antigen bindingdomain”, and as such the term “target” encompasses an “antigen.”

So-called “first-generation” CARs had a targeting domain and a CD3ξsignal transduction domain. So-called “second generation” CARs furtherincluded a co-stimulatory domain, such as a CD28 or 4-1BB domain.So-called “third generation” CARs comprise multiple co-stimulatorydomains. So-called “fourth generation” CARs, also referred to as“TRUCKS” are engineered to release a transgenic cytokine upon CARsignaling.

Chimeric antigen receptors (“CARs”) include the following elements: (1)an optional signal peptide, (2) a target binding domain, (3) an optionalhinge region; (4) a transmembrane region; (5) an intracellular domaincomprising a signal transduction domain. Optionally, the CAR can includeany of: a CD3ζ signal transduction domain, an Fc receptor signaltransduction domain, a co-stimulatory (signal transduction) domain. Thatis, these optional elements can be included in addition to or instead ofother optional elements. The target binding domain is heterologous to atleast one of the other domains. That is, the target binding domain doesnot naturally occur on a T cell receptor, or is not in the same proteinas at least one of the other domains.

The “target binding domain” provides binding specificity to the CAR. The“signal peptide” guides the polypeptide through the cell membrane. Thetarget binding domain can bind to a domain of an antibody that binds tothe target antigen for a so-called “Universal CAR”. The “hinge region”is a flexible connector region, e.g., a natural or syntheticpolypeptide, or any other type of molecule, providing structuralflexibility and spacing to flanking polypeptide regions. The“transmembrane domain” is a membrane-spanning protein domain, typicallyhydrophobic. The “signal transduction domain” or “signaling domain”transmits a signal through a signal transduction pathway into the cellupon binding. Such signaling activates an activity of the cell.“Co-stimulatory domains” are accessory signaling domains that furthertransmit signals.

In some embodiments, the CAR comprises:

-   -   (i) an target-binding domain (also referred to herein as an        antigen binding domain) reactive with a citrullinated protein or        citrullinated fragment thereof, such as a VH-VL or VL-VH,        wherein the two variable domains are separated by a flexible        linker of from 15-25 amino acids in length;    -   (ii) a hinge domain;    -   (iii) a co-stimulatory domain; and    -   (iv) an intracellular signaling domain (also referred to herein        as an activation domain). That is, in some embodiments the CAR        comprises an antigen-binding domain fused to a CAR platform        comprising a hinge domain, a transmembrane domain, and an        intracellular domain comprising a costimulatory domain and an        activation domain. The CAR may further include a signal peptide        (also referred to herein as a leader sequence) to direct        expression of the CAR to the surface of a cell, such as a Treg.

In some embodiments, the CAR comprises an antigen-binding domain fusedin frame to a CAR platform comprising the amino acid sequences of SEQ IDNO:15, SEQ ID:17, SEQ ID NO:28, and SEQ ID:19. In other embodiments, theCAR comprises an antigen-binding domain fused in frame to a CAR platformcomprising the amino acid sequences of SEQ ID NO:30, SEQ ID:16, SEQ IDNO:28, and SEQ ID:19. In some embodiments, the CAR comprises anantigen-binding domain fused in frame to a CAR platform comprising theamino acid sequences of SEQ ID NO:30, SEQ ID:16, SEQ ID NO:29, and SEQID:19. In other embodiments, the CAR comprises an antigen-binding domainfused in frame to a CAR platform comprising the amino acid sequences ofSEQ ID NO:30, SEQ ID:16, SEQ ID NO:29, and SEQ ID:19.

A. Signal Peptide

A signal peptide can be any peptide having the function of allowing apolypeptide to traverse a cell membrane. The signal peptide can bederived from CD4, CD8, CD28, TLR or immunoglobulin family of receptors.

For example, the signal peptide can comprise the sequence:

(SEQ ID NO: 18) MLLLVTSLLLCELPHPAFLLIP; or (SEQ ID NO: 23)MALPVTALLLPLALLLHAAR

B. Target Binding Domain 1. Structure

The target binding domain can include any polypeptide comprising atarget binding function, e.g., an antibody as defined herein. In oneembodiment, the target binding domain can comprise an antibody formretaining antigen binding activity as defined herein. In one embodiment,the target binding domain can comprise a single chain antibody (scFV).The scFv can be connected to the transmembrane domain via a hinge domainwhose length, flexibility and origin provides variability in the CAR'sdesign, and can, along with the transmembrane domain, contribute to theinteraction with antigen, building of the immunologic synapse and impactthe CAR's association with additional proteins needed to impart a robustactivation signal.

2. Targets/Antigens

Chimeric antigen receptors disclosed herein comprise a target bindingdomain (also referred to herein as an antigen-binding domain) that bindsto citrullinated antigens, e.g., those found in the synovium of subjectswith rheumatoid arthritis. In particular, the target binding domain canbind to one or more of (i) citrullinated vimentin, (ii) citrullinatedfilaggrin, (iii) citrullinated fibrinogen and (iv) citrullinatedpeptides thereof. In some embodiments, the target binding domain canbind to a citrullinated peptide fragment of (i)-(iii), wherein thepeptide fragment is at least of 10 amino acids in length, e.g. of atleast 12 amino acids, at least 14 amino acids or at least 16 amino acidsin length. In some embodiments, the target binding domain further bindsto tenascin C. In some embodiments, the target binding domain can bindto two or more of (i) citrullinated vimentin, (ii) citrullinatedfilaggrin, (iii) citrullinated fibrinogen, and (iv) tenascin C, orcitrullinated peptide fragments thereof.

In some embodiments, the target domain is one or more citrullinatedpeptides selected from the following sequences:

(SEQ ID NO: 24) ST(Cit)SVSSSSY(Cit)(Cit)MFGG; (SEQ ID NO: 25)VYAT(Cit)SSAV(Cit)L(Cit)SSV; (SEQ ID NO: 26)(Cit)PAPPPISGGGY(Cit)A(Cit); (SEQ ID NO: 27) SHQEST(Cit)GRSRGRSGRSGS

In some embodiments, the antigen binding domain binds to one or morecitrullinated peptides, but does not bind to non-citrullinatedcounterparts. In some embodiments, the antigen binding domain binds to acitrullinated vimentin peptide set forth as SEQ ID NO:24, but notSTRSVSSSSYRRMFGG (SEQ ID NO:45). In some embodiments, the antigenbinding domain binds to a citrullinated vimentin peptide set forth asSEQ ID NO:25, but not VYATRSSAVRLRSSV (SEQ ID NO:46). In someembodiments, the antigen binding domain binds to a citrullinatedfibrinogen peptide set forth as SEQ ID NO:26, but not RPAPPPISGGGYRAR(SEQ ID NO:47). In some embodiments, the antigen binding domain binds toa citrullinated filaggrin peptide set forth as SEQ ID NO:27, but notSHQESTRGRSRGRSGRSGS (SEQ ID NO:48).

The target binding domain can comprise sequences from an antibody VH andVL domain. In some embodiments, the target binding domain can comprisesequences from heavy chain only antibody or antibody fragment havingonly two VH domains. This includes particular CDR sets from VH and VLdomains. In some embodiments the target binding domain comprises theCDRs from the VH domains of SEQ ID NO:1 or SEQ ID NO:2. In someembodiments the target binding domain comprises the CDRs from the VLdomains of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments the targetbinding domain comprises the CDRs from VH and VL domains of SEQ ID NO:1and SEQ ID NO:3, respectively. In some embodiments the target bindingdomain comprises the CDRs from VH and VL domains of SEQ ID NO:1 and SEQID NO:4, respectively. In some embodiments the target binding domaincomprises the CDRs from VH and VL domains of SEQ ID NO:2 and SEQ IDNO:3, respectively. In some embodiments the target binding domaincomprises the CDRs from VH and VL domains of SEQ ID NO:2 and SEQ IDNO:4, respectively.

In some embodiments the target binding domain comprises thecomplementarity-determining regions (CDRs) from VH and VL domains of SEQID NO:1 (SBT01 VH (M)) and SEQ ID NO:4 (SBT01 VL (G)). In someembodiments, the VH domain of the target-binding domain comprises aVH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2comprising the amino acid sequence of SEQ ID NO:34, and a VH-CDR3comprising the amino acid sequence of SEQ ID NO:36, and the VL domain ofthe target-binding domain comprises a VL-CDR1 comprising the amino acidsequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequenceof SEQ ID NO:41, and a VL-CDR3 comprising the amino acid sequence of SEQID NO:43.

Sequence Region SEQ ID Fragment Residues Length HFR1 NO: 31HLHLQESGPGLVKPS  1-30 30 ETLSLTCTVSGGSIN CDR-H1 NO: 32 DTTYYWG 31-37 7HFR2 NO: 33 WIRQPPGKGLEWIG 38-51 14 CDR-H2 NO: 34 SIYYRGNTHYNSSL 52-6716 RS HFR3 NO: 35 RVTMSVDTSKNRFS 68-99 32 LKVTSVTAADTAVY YCAR CDR-H3NO: 36 LDPFDY 100-105 6 HFR4 NO: 37 WGRGTLVTVSS 106-116 11

Sequence Region SEQ ID Fragment Residues Length LFR1 NO: 38SYVLTQPPSVSVA  1-22 22 PGKTARITC CDR-L1 NO: 39 GGNNIGSKSVH 23-33 11 LFR2NO: 40 WYQQKPGQAPVL 34-48 15 VIY CDR-L2 NO: 41 YDSDRPS 49-55 7 LFR3NO: 42 GIPERFSGSNSGNT 56-87 32 ATLTISRVEAGDEA DYYC CDR-L3 NO: 43QVWDSSSDHQV 88-98 11 LFR4 NO: 44 FGTGTKVTV  99-108 11

In certain embodiments, the target binding domain comprises VH sequencesselected from:

(1) SBT01 VH (M) (SEQ ID NO: 1) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS; (2) SBT01 VH (G) (SEQ ID NO: 2)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLDPFDYWGRGTLVTVSS;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences, provided target binding domain binds to a        citrullinated antigen as described herein.

The target binding region can comprise VL sequences selected from:

(1) SBT01 VL (M) (SEQ ID NO: 3) SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAG DDADYFCQVYDRKTDHQVFGPGTTVTVL;(2) SBT01 VL (G) (SEQ ID NO: 4) SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAG DEADYYCQVWDSSSDHQVFGTGTKVTVL;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences, provided target binding domain binds to a        citrullinated antigen as described herein.

In another embodiment, the antigen binding region comprises an scFVcomprising one or more VH domains comprising an amino acid sequence ofVH domains of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, theantigen binding region comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO:1 or SEQ ID NO:2, provided the scFV domain binds to acitrullinated antigen as described herein.

In some embodiments, the scFV comprises the VL domains of SEQ ID NO:3 orSEQ ID NO:4. In some embodiments, the antigen binding region comprisesan scFV comprising an amino acid sequence having at least any of 80%,85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO: 3 or SEQ ID NO:4,provided the scFV domain binds to a citrullinated antigen as describedherein.

In some embodiments, the scFV domain comprises the VH and VL domains ofSEQ ID NO:1 and SEQ ID NO:3, respectively. In some embodiments, theantigen binding region comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 1 or SEQ ID NO:3, provided the scFV domain binds toa citrullinated antigen as described herein. In some embodiments, alinker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 isplaced between the VH and VL domains.

In some embodiments, the scFV domain comprises the VH and VL domains ofSEQ ID NO:1 and SEQ ID NO:4, respectively. In some embodiments, theantigen binding region comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 1 and SEQ ID NO:4, provided the scFV domain binds toa citrullinated antigen as described herein. In some embodiments, alinker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 isplaced between the VH and VL domains.

In some embodiments, the scFV domain comprises the VH and VL domains ofSEQ ID NO: 2 and SEQ ID NO:3, respectively. In some embodiments, theantigen binding region comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 2 and SEQ ID NO: 3, provided the scFV domain bindsto a citrullinated antigen as described herein. In some embodiments, alinker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 isplaced between the VH and VL domains.

In some embodiments, the scFV domain comprises the VH and VL domains ofSEQ ID NO:2 and SEQ ID NO:4, respectively. In some embodiments, theantigen binding region comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 2 and SEQ ID NO: 4, provided the scFV domain bindsto a citrullinated antigen as described herein. In some embodiments, alinker selected from SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 isplaced between the VH and VL domains.

In another embodiment, the antigen binding region comprises an scFVcomprising an amino acid sequence selected from:

SBT01G-VHVL-GGGSx3 Linker-pSB_0149 (SEQ ID NO: 5)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS GGGG SGGGGSGGGGSSYVLTQPPSVSVAPGKTARITCGGNNIGS KSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (GGGSx3 linker underlined);SBT01G-VHVL-Whitlow 218 Linker-pSB_0158 (SEQ ID NO: 6)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS GSTS GSGKPGSGEGSTKGSYVLTQPPSVSVAPGKTARITCGGNN IGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVT VLR(Whitlow 218 Linker underlined); SBT01G-VHVL-AB pur Linker-pSB_0159(SEQ ID NO: 7) HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCARLDPFDYWGRGTLVTVSS ASSG GSTSGSGKPGSGEGSSGSARSYVLTQPPSVSVAPGKTARI TCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFG TGTKVTVLR(AB pur Linker underlined);or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences, provided target binding domain binds to a        citrullinated antigen as described herein.

For example, the linker can comprise the sequence:

(SEQ ID NO: 20) GGGGSGGGGSGGGGS GGGSx3 Linker; or (SEQ ID NO: 21)GSTSGSGKPGSGEGSTKG Whitlow 218 Linker; or (SEQ ID NO: 22)ASSGGSTSGSGKPGSGEGSSGSAR AB pur Linker.

Optionally, any of the foregoing sequences can include the CDR sets fromthe VH and VL domains described above.

C. Hinge Region

In some embodiments, the hinge region of the disclosed CARs can beselected from the CD8, CD4, or CD28 extracellular domain, the Fc regionof an IgG1 antibody, or the extracellular domain of any of the TLRreceptors as is known to one of skill in the art and can be found in theGenBank database.

For example, the hinge region can comprise the sequence:

(SEQ ID NO: 30) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (CD28); or(SEQ ID NO: 15) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (CD8);or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned hinge        region sequences.

D. Transmembrane Domain

The transmembrane domain can comprise a transmembrane domain of animmunoglobulin family receptor, such as CD8. The intracellular domaincan be selected from any membrane-spanning molecule on a T cell. Forexample, the transmembrane (TM) domain of the disclosed CAR can comprisethe TM domain of CD2, CD3, CD16, CD32, CD64, CD28, CD247, 4-1BBL, CD4,or CD8.

For example, the transmembrane domain can comprise the sequence:

(SEQ ID NO: 16) FWVLVVVGGVLACYSLLVTVAFIIFWV; or (SEQ ID NO: 17)IYIWAPLAGTCGVLLLSLVITLYC;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned hinge        region sequences.

E. Signal Transduction Domain 1. CD3ζ Signal Transduction Domain

In some embodiments, the signal transduction domain comprises a CD3ζsignaling domain. The CD3ζ signaling domain of the disclosed CARmolecule can comprise a CD3ζ amino acid sequence, e.g., a signaltransduction domain of CD3 zeta.

For example, the CD3ζ signal transduction domain can comprise thesequence:

(SEQ ID NO: 19) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned CD3 zeta        signal transduction domain sequence.

For example, the CD3 zeta signal transduction domain can include aminoacids 21-163, 31-142, 68-89 and/or 138-158 of the sequence shown in SEQID NO:19, or functional variants thereof (e.g., with 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or 10-20 amino acid substitutions, deletions, oradditions).

2. Fc Receptor Signal Transduction Domain

In some embodiments, the signal transduction domain comprises an Fcsignaling domain. The Fc signaling domain can be any one of theFc-alpha, Fc-gamma, Fc-epsilon, Fc-mu, and Fc-delta receptors. Forexample, the Fc receptor signaling domain can comprise amino acidsinvolved in interaction with Src (e.g., Fgr, Fyn, Hck, Lyn, Yes, andSrc) and ZAP-70 family kinases, e.g., one or more ITAM domains (see,e.g., Sanchez-Mejorada et al. (1998) J. Leukocyte Biol. 63:531;Garcia-Garcia et al. (2002) J. Leukocyte Biol. 72:1092). In someembodiments, the Fc receptor signal transduction domain includes atleast one ITAM domain, e.g., from any one of the Fc-alpha, Fc-gamma,Fc-epsilon, Fc-mu, and Fc-delta receptors, or substantially identicalthereto.

Sequences also can be found as follows:

Molecule GenBank No. CD3ζ NP_000725.1 Fc gamma receptor family (CD16)NP_000560.5 NP_001231682.1 Fc gamma receptor family (CD32) AAH20823.1AAH19931.1 AAI48274.1 AAI37398.1 Fc gamma receptor family (CD64)AAI60240.1 AAH32634.1 AAI56865.1

F. Co-Stimulatory Domain

The CARs of this disclosure can include one or more co-stimulatorydomains in addition to a signal transduction domain of CD3ζ or an Fcreceptor. Co-stimulatory domains can be derived from, for example, CD28,4-1BB, CD2, CD27, CD30, OX40, CD40, PD-1, PD-Li, PD-L2, ICOS, LFA-1,CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CO80), B7-2 (CD86), B7-H3, B7-H4and others. The CAR constructs can contain two or more co-stimulatorysignaling domains (e.g., CD28 and 4-1BB).

The co-stimulatory domain or domains can be positioned between thesignal transduction domain and the transmembrane region.

In certain embodiments, a CD28 co-stimulatory domain can comprise thesequence:

(SEQ ID NO: 29) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned sequence.

In certain embodiments, a 41BB co-stimulatory domain can comprise thesequence:

(SEQ ID NO: 28) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned sequence.

III. Nucleic Acids A. Nucleic Acids Encoding CARs

Disclosed herein are nucleic acid molecules (polynucleotides) comprisinga nucleotide sequence that encodes a CAR of this disclosure. The nucleicacid of the disclosed CAR can be in the form of DNA or in the form ofRNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can bedouble-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand. RNA includes mRNA,siRNA, sRNA, ssRNA and so on.

For example, nucleic acids can comprise nucleotide sequences that encodethe polypeptides of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4. In certain embodiments, the nucleotide sequences encoding VHdomains comprise:

(1) SBT01 VH (M) (SEQ ID NO: 8)CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGC,(2) SBT01 VH (G) (SEQ ID NO: 9)CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGC;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences.

In certain embodiments, the nucleotide sequences encoding VL domainscomprise:

(1) SBT01 VL (M) (SEQ ID NO: 10)TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGAGACGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATGTCAACTGGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATCTTCTTTGATACCAGACGGCCCTCAGGGATCCCGGAGCGATTCTCTGGCTCCAGGTCTGAGGACACGGCCAACCTGACCATCACCAGGGTCGAGGCCGGGGATGACGCCGACTATTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA; (2) SBT01 VL (G) (SEQ ID NO: 11)TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA;or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences.

In another embodiment, the nucleic acid molecule encoding VH domainscomprise SEQ ID NO: 8 or SEQ ID NO:9. In some embodiments, the nucleicacid molecule encoding VH domains comprise a nucleic sequence having atleast any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:8or SEQ ID NO:9, provided the scFV domain binds to a citrullinatedantigen as described herein.

In some embodiments, the nucleic acid molecule encoding VL domainscomprise SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, thenucleic acid molecule encoding VL domains comprise a nucleic sequencehaving at least any 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ IDNO: 10 or SEQ ID NO: 11, provided the scFV domain binds to acitrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding an scFV domaincomprise SEQ ID NO:8 and SEQ ID NO:10. In some embodiments, the nucleicacid molecule encoding the scFV comprises an amino acid sequence havingat least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:8 or SEQ ID NO:10, provided the scFV domain binds to a citrullinatedantigen as described herein.

In some embodiments, the nucleic acid molecule encoding an scFV domaincomprise SEQ ID NO: 8 and SEQ ID NO: 11. In some embodiments, thenucleic acid molecule encoding the scFV comprises an amino acid sequencehaving at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQID NO: 8 or SEQ ID NO:11, provided the scFV domain binds to acitrullinated antigen as described herein.

In some embodiments, the nucleic acid molecule encoding an scFV domaincomprise SEQ ID NO: 9 and SEQ ID NO:10. In some embodiments, the nucleicacid molecule encoding the scFV comprises an amino acid sequence havingat least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:9 or SEQ ID NO: 10, provided the scFV domain binds to a citrullinatedantigen as described herein.

In some embodiments, the nucleic acid molecule encoding an scFV domaincomprise SEQ ID NO: 9 and SEQ ID NO:11. In some embodiments, the nucleicacid molecule encoding the scFV comprises an amino acid sequence havingat least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ ID NO:9 or SEQ ID NO: 11, provided the scFV domain binds to a citrullinatedantigen as described herein.

In another embodiment, the nucleic acid molecule encodes an scFvmolecule and has a nucleotide sequence:

SBT01G-VHVL-GGGSx3 Linker-pSB_0149 (SEQ ID NO: 12)CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATC TTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC (GGGSx3 linker underlined);or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned sequence.

In another embodiment, the nucleic acid molecule encodes an scFvmolecule and has a nucleotide sequence:

SBT01G-VHVL-Whitlow 218 Linker-pSB_0158 (SEQ ID NO: 13)CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGCGGAAGCACGAGTGGTTCAGGCAAACCGGGTTCCGGTGAAGGTTC AACAAAAGGTTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC (Whitlow 218 Linker underlined);or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned sequence.

In another embodiment, the nucleic acid molecule encodes an scFvmolecule and has a nucleotide sequence:

SBT01G-VHVL-AB pur Linker-pSB_0159 (SEQ ID NO: 14)CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGCGCCTCTAGCGGGGGGAGCACATCAGGAAGCGGCAAGCCCGGTAGCGGCGAAGGCTCCTCTGGCAGCGCCCGC TCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGC (AB pur Linker underlined);or

-   -   a sequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%,        99% or 99.5% sequence identify with the aforementioned        sequences.

Optionally, these can include sequences encoding the CDR sets from theVH and VL domains described herein.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, thepolynucleotide variants contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded CAR polypeptide. In some embodiments, thepolynucleotide variants contain alterations that do not produce anychanges in the amino acid sequence. In some embodiments, polynucleotidevariants contain “silent” substitutions due to the degeneracy of thegenetic code. Polynucleotide variants can be produced for a variety ofreasons, for example, to optimize codon expression for a particularhost.

In some embodiments, the polynucleotides as described herein areisolated.

Polynucleotides encoding CARs be isolated molecules, or can be includedwithin a vector, such as a plasmid, a cosmid, an artificial chromosomeor a virus. Such vectors can be used to transfect target cells.

B. Expression Constructs and Vectors

Polynucleotides encoding CARs of this disclosure can include regulatoryelements operatively linked with a nucleotide sequence encoding the CAR.For example, a polynucleotide can include one or more transcriptionregulatory elements, such as promoters or enhancers, which, when thepolynucleotide is present in a cell, cause the sequence encoding the CARto be expressed within the cell.

Nucleic acids disclosed herein can be incorporated into vectors capableof transfecting cells. Such vectors include, without limitation, viralvectors plasmids and microvesicles, e.g., liposomes. Exemplary viralvectors adenoviral vectors Ad, AAV, lentivirus, and vesicular stomatitisvirus (VSV) and retroviruses. Lentiviruses are a genus of theRetroviridae family and include HIV, SIV, and FIV. Lentiviruses candeliver a large quantities of genetic material into the DNA of the hostcell. They are able to infect non-dividing cells.

IV. Cells

In some embodiments, the recombinant (host) cell having the nucleic acidmolecule encoding the disclosed CAR wherein the nucleic acid moleculecan further comprises an expression control sequence operatively linkedwith the nucleotide sequence encoding the CAR. The assembled CAR (bysynthesis, site-directed mutagenesis or another method, as is known toone of skill in the art), the nucleic acid molecule encoding thedisclosed CAR can be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe disclosed CAR in a desired host. Correct assembly can be confirmedby nucleotide sequencing, restriction mapping, and/or expression of theCAR polypeptide in a suitable host. As is well known in the art, inorder to obtain high expression levels of a transfected gene in a host,the gene must be operatively linked to transcriptional and translationalexpression control sequences that are functional in the chosenexpression host.

This disclosure also provides cells (e.g., recombinant cells) comprisingnucleic acid molecules encoding CARs and/or expressing CARs.

The nucleic acid molecule encoding the disclosed CAR can be delivered toa host cell, including but not limited to a T cell, B cell, myeloidprogenitor, macrophage, and so on, by a plasmid or a viral vector as isknown to one of skill in the art. The resulting recombinant (host) cellcan include but is not limited to a T-cell, a CD4 T-cell, a Treg cell, aCD8 alpha T-cell, CD8 beta T cell, T helper cell, granulocyte(neutrophils, basophils, eosinophils), megakaryocytes, monocyte,macrophage and a dendritic cell, a T memory stem cell as well as cellsexpressing MHC class I or class II as is known to one of skill in theart. In some embodiments, the recombinant (host) cell having the nucleicacid molecule encoding the disclosed CAR can be a myeloid progenitorcell selected from the group consisting of a common myeloid progenitor,a granulocyte macrophage progenitor, a megakaryocyte erythrocyteprogenitor, a granulocyte progenitor and a monocyte progenitor as isknown to one of skill in the art. In some embodiments, the myeloid cellis an autologous or allogeneic cell.

In some embodiments, the cells expressing the CARs of this disclosureare Treg cells. “Regulatory T cells,” or “T_(reg) cells,” are cellsbelonging to a specialized subpopulation of T cells that act to suppressimmune response, thereby maintaining homeostasis and self-tolerance.T_(reg)s are able to inhibit T cell proliferation and cytokineproduction and play a critical role in preventing autoimmunity. T_(reg)sare characterized by expression of FoxP3. Surface markers for T_(reg)sinclude CD4, CD25high (high molecular density) and CD127low (lowmolecular density). Mouse and human Tregs express GITR/AITR, and CTLA-4.Human CD4+FoxP3+ Treg cells can be divided into three sub-populations:(1) CD45RA+CD25+FoxP3l0w resting Treg cells, (2)CD45RO+CD25highFoxP3high activated Treg cells, and (3) proinflammatorycytokine-producing CD45RO+CD25+FoxP3low nonsuppressive effector T cells(Teffs).

The cells to be transformed with the nucleic acids disclosed herein canbe cells taken from a subject into whom the recombinant cells are to beadministered. In this way, issues of an allogeneic immune response canbe mitigated.

Cells can be expanded ex vivo before administration to a subject.

Treg cells into which nucleic acids expressing the CARs of thisdisclosure have been incorporated can express these CARs and be used inthe methods described herein to treat rheumatoid arthritis.

Also, the proteins produced by a transformed/recombinant host can bepurified according to any suitable method. Such methods includechromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for protein purification. Affinity tags such ashexa-histidine, maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. In someembodiments, proteins can also be physically characterized using suchtechniques as proteolysis, high performance liquid chromatography(HPLC), nuclear magnetic resonance and x-ray crystallography.

V. Compositions

Also disclosed are pharmaceutical compositions comprising a recombinantcell having a nucleic acid molecule encoding the disclosed CARpolypeptide and/or expressing the disclosed CAR polypeptide, and apharmaceutically acceptable carrier, as well as methods of use in thetreatment rheumatoid arthritis.

As used herein, the term “pharmaceutical composition” refers to acomposition comprising a pharmaceutical compound (e.g., a drug or arecombinant Treg cell as described herein) and a pharmaceuticallyacceptable carrier.

As used herein, the term “pharmaceutically acceptable” refers to acarrier that is compatible with the other ingredients of apharmaceutical composition and can be safely administered to a subject.The term is used synonymously with “physiologically acceptable” and“pharmacologically acceptable”. Pharmaceutical compositions andtechniques for their preparation and use are known to those of skill inthe art in light of the present disclosure. For a detailed listing ofsuitable pharmacological compositions and techniques for theiradministration one may refer to texts such as Remington's PharmaceuticalSciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman's ThePharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University ofthe Sciences in Philadelphia (eds.), “Remington: The Science andPractice of Pharmacy,” Lippincott Williams & Wilkins, 2005; andUniversity of the Sciences in Philadelphia (eds.), “Remington: ThePrinciples of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

Pharmaceutically acceptable carriers will generally be sterile, at leastfor human use. A pharmaceutical composition will generally compriseagents for buffering and preservation in storage, and can includebuffers and carriers for appropriate delivery, depending on the route ofadministration. Examples of pharmaceutically acceptable carriersinclude, without limitation, normal (0.9%) saline, phosphate-bufferedsaline (PBS) Hank's balanced salt solution (HBSS) and multipleelectrolyte solutions such as PlasmaLyte ATM (Baxter).

Pharmaceutical compositions can be formulated for any route ofadministration, including mucosal (e.g., nasal, sublingual, vaginal,buccal, or rectal), parenteral (e.g., subcutaneous, intravenous,intramuscular, or intraarterial injection, either bolus or infusion),oral, or transdermal.

Injectable (e.g., intravenous) compositions can comprise a solution ofthe composition suspended in an acceptable carrier, such as an aqueouscarrier. Any of a variety of aqueous carriers can be used, e.g., water,buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5%dextrose, and the like, and may include glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc. Often, normalbuffered saline (135-150 mM NaCl) will be used. The compositions cancontain pharmaceutically acceptable auxiliary substances to approximatephysiological conditions, such as pH adjusting and buffering agents,tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc. In some embodiments, thecomposition can be formulated in a kit for intravenous administration.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Injection solutions and suspensions can also beprepared from sterile powders, granules, and tablets. In the practice ofthe present invention, compositions can be administered, for example, byintravenous infusion, topically, intraperitoneally, intravesically, orintrathecally. The formulations of compositions can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials.

Cells can be cryopreserved. Cryopreservation can include formulatingcells with a cryopreservation agent, such as DMSO. Commerciallyavailable media include, for example, CryoStor® and pZerve®, availablefrom Millipore Sigma.

Compositions can be formulated as dosage forms for administration. Theterm “dosage form” refers to the particular format of thepharmaceutical, and depends on the route of administration. Examples ofdosage forms include, but are not limited to: dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or awater-in-oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. The dose will vary depending on a number of factors,including frequency of administration; size and tolerance of theindividual; severity of the condition; risk of side effects; the routeof administration; and the imaging modality of the detectable label (ifpresent). One of skill in the art will recognize that the dose can bemodified depending on the above factors or based on therapeuticprogress.

The pharmaceutical preparation can be packaged or prepared in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g.,according to the dose of the therapeutic agent or concentration of thecomposition. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation. The compositioncan, if desired, also contain other compatible therapeutic agents.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising the complementarity-determining regions (CDRs)from the VH domains of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments,the compositions of the invention comprise a recombinant cell having anucleic acid molecule encoding a CAR polypeptide comprising the CDRsfrom the VL domains of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments,the compositions of the invention comprise a recombinant cell having anucleic acid molecule encoding a CAR polypeptide comprising the CDRsfrom VH and VL domains of SEQ ID NO:1 and SEQ ID NO:3. In someembodiments, the compositions of the invention comprise a recombinantcell having a nucleic acid molecule encoding a CAR polypeptidecomprising the CDRs from VH and VL domains of SEQ ID NO:1 and SEQ IDNO:4. In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising the CDRs from VH and VL domains of SEQ ID NO:2and SEQ ID NO:3. In some embodiments, the compositions of the inventioncomprise a recombinant cell having a nucleic acid molecule encoding aCAR polypeptide comprising the CDRs from VH and VL domains of SEQ IDNO:2 and SEQ ID NO:4. In some embodiments, the cell is a T-cell, a CD4T-cell, a Treg cell, a CD8 alpha T-cell, CD8 beta T cell, T helper cell,granulocyte (neutrophils, basophils, eosinophils), megakaryocytes,monocyte, macrophage and a dendritic cell, or a T memory stem cell. Insome embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising the CDRs from VH and VL domains of SEQ ID NO:1(SBT01 VH (M)) and SEQ ID NO:4 (SBT01 VL (G)). In some embodiments, theVH domain of the CAR polypeptide comprises a VH-CDR1 comprising theamino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:36, and the VL domain of CAR polypeptide comprisesa VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2comprising the amino acid sequence of SEQ ID NO:41, and a VL-CDR3comprising the amino acid sequence of SEQ ID NO:43. In some embodiments,the cell is a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8beta T cell, T helper cell, granulocyte (neutrophils, basophils,eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell,or a T memory stem cell. In some embodiments, the cell is a Treg cell.

In another embodiment, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising one or more VH domainscomprising the amino acid sequence of VH domains of SEQ ID NO:1 or SEQID NO:2. In some embodiments, the nucleic acid molecule encoding a CARpolypeptide comprises an scFV comprising an amino acid sequence havingat least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQ IDNO:1 or SEQ ID NO:2, provided the scFV domain binds to a citrullinatedantigen as described herein. In some embodiments, the cell is a T-cell,a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8 beta T cell, T helpercell, granulocyte (neutrophils, basophils, eosinophils), megakaryocytes,monocyte, macrophage and a dendritic cell, or a T memory stem cell. Insome embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising the VL domains of SEQ ID NO:3or SEQ ID NO:4. In some embodiments, the nucleic acid molecule encodinga CAR polypeptide comprises an scFV comprising an amino acid sequencehaving at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% of SEQID NO: 3 or SEQ ID NO:4, provided the scFV domain binds to acitrullinated antigen as described herein. In some embodiments, the cellis a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8 beta Tcell, T helper cell, granulocyte (neutrophils, basophils, eosinophils),megakaryocytes, monocyte, macrophage and a dendritic cell, or a T memorystem cell. In some embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising the VH and VL domains of SEQID NO:1 and SEQ ID NO:3. In some embodiments, the nucleic acid moleculeencoding a CAR polypeptide comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 1 or SEQ ID NO:3, provided the scFV domain binds toa citrullinated antigen as described herein. In some embodiments, thecell is a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8beta T cell, T helper cell, granulocyte (neutrophils, basophils,eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell,or a T memory stem cell. In some embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising the VH and VL domains of SEQID NO:1 and SEQ ID NO:4. In some embodiments, the nucleic acid moleculeencoding a CAR polypeptide comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 1 and SEQ ID NO:4, provided the scFV domain binds toa citrullinated antigen as described herein. In some embodiments, thecell is a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8beta T cell, T helper cell, granulocyte (neutrophils, basophils,eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell,or a T memory stem cell. In some embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising the VH and VL domains of SEQID NO: 2 and SEQ ID NO:3. In some embodiments, the nucleic acid moleculeencoding a CAR polypeptide comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 2 and SEQ ID NO: 3, provided the scFV domain bindsto a citrullinated antigen as described herein. In some embodiments, thecell is a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8beta T cell, T helper cell, granulocyte (neutrophils, basophils,eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell,or a T memory stem cell. In some embodiments, the cell is a Treg cell.

In some embodiments, the compositions of the invention comprise arecombinant cell having a nucleic acid molecule encoding a CARpolypeptide comprising an scFV comprising the VH and VL domains of SEQID NO:2 and SEQ ID NO:4. In some embodiments, the nucleic acid moleculeencoding a CAR polypeptide comprises an scFV comprising an amino acidsequence having at least any of 80%, 85%, 90%, 95%, 97%, 98%, 99% or99.5% of SEQ ID NO: 2 and SEQ ID NO: 4, provided the scFV domain bindsto a citrullinated antigen as described herein. In some embodiments, thecell is a T-cell, a CD4 T-cell, a Treg cell, a CD8 alpha T-cell, CD8beta T cell, T helper cell, granulocyte (neutrophils, basophils,eosinophils), megakaryocytes, monocyte, macrophage and a dendritic cell,or a T memory stem cell. In some embodiments, the cell is a Treg cell.

VI. Methods of Use

T cells, and in particular, Treg cells, that express the CARs disclosedherein are useful in the treatment of rheumatoid arthritis. Methods ofuse comprise administering an effective amount a pharmaceuticalcomposition of this disclosure to a subject in need thereof, e.g., asubject suffering from rheumatoid arthritis.

As used herein, the term “subject” refers to an individual animal. Theterm “patient” as used herein refers to a subject under the care orsupervision of a health care provider such as a doctor or nurse.Subjects include mammals, such as humans and non-human primates, such asmonkeys, as well as dogs, cats, horses, bovines, rabbits, rats, mice,goats, pigs, and other mammalian species. Subjects can also includeavians. A patient can be an individual that is seeking treatment,monitoring, adjustment or modification of an existing therapeuticregimen, etc. The term “rheumatoid arthritis subject” refers to anindividual that has been diagnosed with rheumatoid arthritis. Rheumatoidarthritis patients can include individuals that have not receivedtreatment, are currently receiving treatment, have had treatment, andthose that have discontinued treatment.

As used herein, the terms “effective amount,” “effective dose,” and“therapeutically effective amount,” refer to an amount of an agent thatis sufficient to generate a desired response, such as reduce oreliminate a sign or symptom of a condition or ameliorate a disorder. Insome examples, an “effective amount” is one that treats (includingprophylaxis) one or more symptoms and/or underlying causes of any of adisorder or disease and/or prevents progression of a disease. Forexample, for the given parameter, a therapeutically effective amountwill show an increase or decrease of therapeutic effect at least any of5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol.

The pharmaceutical composition can be administered by any suitableroute, including but not limited to intravenous, subcutaneous,intramuscular or intraperitoneal routes. An example of administration ofa pharmaceutical composition includes storing the composition at 10mg/ml in sterile isotonic aqueous saline solution for injection at 4°C., and diluting it in either 100 ml or 200 ml 0.9% sodium chloride forinjection prior to administration to the patient. The pharmaceuticalcomposition is administered by intravenous infusion over the course of 1hour at a dose of between 0.2 and 10 mg/kg. In other embodiments, thepharmaceutical composition is administered by intravenous infusion overa period of between 15 minutes and 2 hours. In still other embodiments,the administration procedure is via sub-cutaneous bolus injection.

The dose of the composition is chosen in order to provide effectivetherapy for the patient and is in the range of less than 0.1 mg/kg bodyweight to about 25 mg/kg body weight or in the range 1 mg-2 g perpatient. In some cases, the dose is in the range 1-100 mg/kg, orapproximately 50 mg-8000 mg/patient. The dose may be repeated at anappropriate frequency which may be in the range once per day to onceevery three months, depending on the pharmacokinetics of the composition(e.g., half-life of the composition in the circulation) and thepharmacodynamic response (e.g., the duration of the therapeutic effectof the composition). In some embodiments, the in vivo half-life ofbetween about 7 and about 25 days and composition dosing is repeatedbetween once per week and once every 3 months.

Administration can be periodic. Depending on the route ofadministration, the dose can be administered, e.g., once every 1, 3, 5,7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6months). In some cases, administration is more frequent, e.g., 2 or 3times per day. The patient can be monitored to adjust the dosage andfrequency of administration depending on therapeutic progress and anyadverse side effects, as will be recognized by one of skill in the art.

Thus, in some embodiments, additional administration is dependent onpatient progress, e.g., the patient is monitored betweenadministrations. For example, after the first administration or round ofadministrations, the patient can be monitored for rate of tumor growth,recurrence (e.g., in the case of a post-surgical patient), or generaldisease-related symptoms such as weakness, pain, nausea, etc.

In certain embodiments, the T cells described herein are administered tothe synovia of the joints of subjects having rheumatoid arthritis. Suchadministration can be by injection directly into the joint.

An exemplary method of this disclosure includes isolating T lymphocytesfrom a biological sample obtained from the subject. Such T cell can beisolated by immunoaffinity, for example, using solid supportsderivatized and anti-CD4 antibodies. CD4+ T regulatory cells (Treg) canbe separated from non-Treg cells based on their marker profile. Tregcells are CD4+, CD25+, CD127lo. Non Treg cells are: CD4+, CD25+ andCD127+. The isolated Treg cells are then transfected with an expressionvector encoding a chimeric antigen receptor (CAR) of this disclosure.The transfected cells are expanded. The expanded cells are administeredto the subject.

VII. Kits

As used herein, the term “kit” refers to a collection of items intendedfor use together. The kit can optionally include a reference agentand/or instructions for use thereof. A kit can further include ashipping container adapted to hold a container, such as a vial, thatcontains a composition as disclosed herein. A kit can include acontainer that contains within it the collection of items.

Kits of this disclosure can comprise a pharmaceutical composition asdescribed herein, contained in a container, such as a bag or bottle forintravenous administration. Also included in the kit can be a fluidicconduit, such as a plastic tube, with a drip chamber. The drip chambercan communicate through a fluidic conduit with an intravenous needle.The fluidic conduit also can comprise one or more Y-sites and a rollerclamp.

EXAMPLES

Abbreviations: CAR (chimeric antigen receptor); CF (citrullinatedfibrinogen); CFSE (carboxyfluorescein succinimidyl ester); CV(cirtullinated vimentin); EGFR (epidermal growth factor receptor); IN(intranasal); IV (intravenous); LPS (lipopolysaccharide); PAD2(peptidylarginine deiminase 2); PBMC (peripheral blood mononuclearcells); PBS (phosphate buffered saline); RA (rheumatoid arthritis); scFv(single-chain variable fragment); SF (synovial fluid); Teff (effector Tcell); Treg (regulatory T cell); and UTD (untransduced).

Example 1: SBT01G Consistently Performed as well as, or Superior toBVCA1 in a Luciferase System

Jurkat-FF-Luciferase Transduction×50,000 cells per well were plated ineach of 2 flat-bottom 96-well plates in RPMI with 2× protamine sulfate.Viruses were diluted in RPMI such that 100 μl is approx. an MOI of 1.Then 100 μl was added to one column on each 96-well plate. Plates werethen spun and placed in an incubator. Samples were pooled the next day.Transduced cells were resuspended in media and transferred 100 μl to aU-bottom plate in duplicate. The cells were stained with CV-AF488 andCV-AF647 with 1:100 anti-EGFR-PE at 4° C. for 20 mins, washed once, thenanalyzed by Novocyte. Samples were then scaled up into 6-well plates byadding 3 ml fresh media

Plate Coating—Citrullinated vimentin (CV) was first diluted 1:100 into 4ml PBS. 5 serial 4-dilutions were made by transferring 1 ml into 3 ml.100 μl was then added to 96-well plates. Plates were placed at 4° C. tocoat overnight.

Luciferase Assay—Normalized transduced cells were pelleted andresuspended in 1 ml RPMI. The CV-coated plates were washed three timeswith PBS. 50 μl of cells were added to the plates. 25 μl of 3× PMA/Ionowere added, and the plates were placed in the 37° C. incubator.Approximately 24 hours later, the luciferase plates were read by adding75 μl BioGlo reagent, incubating 2-3 mins in the dark and reading theplate.

FIG. 1 shows the response of virus transduced Jurkat-FF-Luciferase cellsto different concentrations of full CV coated plates. The results showthat BVCA1and STB01 had the strongest response to plate-bound,full-length CV.

FIG. 2 shows the response of virus transduced Jurkat-FF-Luciferase cellsto different concentrations of soluble CV. The results show that onlyBVCA1 and SBT01G-HL had a dose-dependent response to soluble,full-length CV.

FIG. 3 shows that in an assay with soluble bead-bound-peptide, BVCA1 andSBT01 demonstrated binding specificity to CV.

FIG. 4 shows the response of BVCA1 and SBT01G to plate-bound CV,antibody (V9) captured CV, and soluble CV at different proteinconcentrations. The results show that SBT01G has a stronger response toplate-bound CV and antibody (V9) captured CV, but not soluble CV, thanBVCA1.

Example 2: SBT01 and BVCA1 Respond to Synovial Fluids from RheumatoidArthritis Patients

Jurkat-FF-Luciferase transduction—24×10⁶ Jurkat-FF-luc cells werepelleted and resuspended in 24 ml RPMI containing protamine sulfate andvirus at a MOI of 3, to express CARs of pSB_0147, pSB-0149 and pSB0139.Cells were mixed and 4 ml were aliquoted into each well of a 6-wellplate. Plates were then spun and placed in an incubator overnight. Cellswere pelleted and reseeded in 25 ml RPMT in T75 flasks.

Synovial Fluid Stimulus—Transduced cells were pelleted and resuspendedin RPMI and placed in wells of black/white plates. Synovial fluid (SF)samples from RA patients were thawed, vortexed, and diluted in RPMIbefore being added to plates containing transduced cells. Cells werecultured with SF at 37° C.

Luciferase Assay—Approximately 24 hours later, 75 μl BioGlo reagent wasadded to each well, and the plates were incubated 2-3 mins in the dark.Luminescence was then read on a plate reader.

Treg isolation and Tissue Culture—Primary human Treg cells were sourcedfrom healthy donors from leukoreduction chamber residuals or leukopaks.Peripheral blood mononuclear cells (PBMC) were isolated by densitygradient centrifugation using Ficoll-Paque Plus. CD25+ cells wereenriched by positive selection. Treg cells were next isolated using FACSby gating for CD4+CD25+CD127lo cells. After isolation, cells werestimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead to cellratio and cultured in RPMI medium, supplemented with 10% FBS,non-essential amino acids, sodium pyruvate, and beta-mercaptoethanolwith recombinant human IL-2 at 300 IU/mL at a density of 0.25-0.3million cells/mL. On day 9 of expansion, fresh CTS Dynabeads TregXpander were added at a 1:1 bead to cell ratio.

Transduction of Primary Tregs—Primary Tregs were transduced with CV-CARconstructs on day 2 of expansion via spin-occulation in the presence ofprotamine sulfate.

Activation of Tregs—CV-CAR-expressing Treg cells were cultured in vitrowith a dilution of synovial fluid samples ranging from 1:5 to 1:160.Treg activation was assessed by measurement of CD71 expression.

Flow Cytometry and FACS Analysis—Activation cultures were collected andcentrifuged at 300×g for 5 min and then resuspended in 1× FlowstainBuffer (Invitrogen) with a viability dye (Invitrogen), anti-EGFR andCD71 surface staining antibody. Tregs were incubated for 30 min at 4°C., then centrifuged and washed with 1× Flow stain Buffer. Stained cellswere fixed with CytoFix (BD Biosciences) then analyzed by flowcytometry.

FIG. 5 shows the response of SBT01G and BVCA1 to synovial fluids from RApatients. The data shows that SBT01G gave stronger responses than BVCA1to synovial fluids from several RA patients.

FIG. 6 shows for synovial fluid samples from Swedish RA patients thattriggered a response, that SBT01G was again stronger than BVCA1.

FIG. 7 shows responses of primary Treg cells transduced with CV-CARs tosynovial fluid from multiple RA patients. The primary Treg cellsexpressing the SBT01G CAR are more sensitive to synovial fluid from RApatients than primary Treg cells expressing the BVCA1 CAR.

Example 3: SBT01 and BVCA1 Response to Citrullinated Fibrinogen

Plate Coating—Citrullinated protein was diluted in PBS and added towells of a black/white isoplate. Plates were placed at 4° C. overnightto coat the plate wells.

Luciferase Assay—Stably-transduced and untransduced (UTD) Jurkat-FF-luccell lines were pelleted and resuspended in RPMI containing 10% FBS. Thecitrullinated protein-coated plates were washed three times with PBS.50,000 cells per well in 75 μl were added the coated plates. 5 μl of15×PMA/Iono in RPMI were added to each well, and the plates were placedin the 37° C. incubator. Approximately 24 hours later, 75 μl BioGloreagent was added to each well, and the plates were incubated 2-3 minsin the dark. Luminescence was then read on a plate reader.

Treg Isolation and Tissue Culture—Primary human Treg cells were sourcedfrom healthy donors from leukoreduction chamber residuals or leukopaks.Peripheral blood mononuclear cells (PBMC) were isolated by densitygradient centrifugation using Ficoll-Paque Plus. CD25+ cells wereenriched by positive selection. Treg cells were next isolated using FACSby gating for CD4+CD25+CD127lo cells and Teff were isolated using FACSgating for CD4+CD25loCD127pos. After isolation, cells were stimulatedwith CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead to cell ratio andcultured in RPMI medium, supplemented with 10% FBS, non-essential aminoacids, sodium pyruvate, and b-mercaptoethanol with recombinant humanIL-2 at 300 IU/mL at a density of 0.25-0.3 million cells/mL. On day 9 ofexpansion fresh CTS Dynabeads Treg Xpander were added at a 1:1 bead tocell ratio.

Transduction of Primary Tregs and Teffs—Primary Tregs and Teff weretransduced with CD19-CAR or CV-CAR constructs on day 2 of expansion viaspin-occulation in the presence of protamine sulfate.

Activation of Tregs—Before start of activation cultures CAR expressingTregs and Teff were labelled with a proliferation dye, CFSE. CD19-CARand CV-CAR-expressing Treg cells were cultured in vitro withplate-coated citrullinated vimentin (CV) or citrullinated fibrinogen(CF) ranging in dose from 30 ng/ml to 10 μg/ml. Treg activation wasassessed by measurement of percentages of proliferating cells and byCD71 expression.

Flow Cytometry and FACS Analysis—Activation cultures were collected andcentrifuged at 300×g for 5 min and then resuspended in 1× FlowstainBuffer (Invitrogen) with a viability dye (Invitrogen), anti-EGFR andCD71 surface staining antibody. Tregs were incubated for 30 min at 4°C., then centrifuged and washed with 1× Flowstain Buffer. Stained cellswere fixed with CytoFix (BD Biosciences) then analyzed by flowcytometry.

FIG. 8 shows responses of SBT01G and BVCA1 to plate-bound, full-lengthpeptidylarginine deiminase 2 (PAD2)-citrullinated fibrinogen. Theresults show that SBT01G, but not BVCA1, is also able to respond toplate-bound, full-length PAD2-citrullinated fibrinogen.

FIG. 9 shows that SBT01G CAR expressed on Teff cells and Treg cellsrespond to citrullinated vimentin (CV) and citrullinated fibrinogen(CF). In contrast, BVCA1 CAR expressed on Teff cells and Treg cellsrespond to CV, but not to CF.

Thus, FIGS. 1-9 show that SBT01G consistently performed as well as, orsuperior to BVCA1 in all assay systems.

Example 4: CV CARS Demonstrate Functional Responses when DifferentPromoters and Linkers are Used

Treg Isolation and Tissue Culture—Primary human Treg cells were sourcedfrom healthy donors from leukoreduction chamber residuals or leukopaks.Peripheral blood mononuclear cells (PBMC) were isolated by densitygradient centrifugation using Ficoll-Paque Plus. CD25+ cells wereenriched by positive selection. Treg cells were next isolated using FACSby gating for CD4+CD25+CD127lo cells. After isolation, cells werestimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead to cellratio and cultured in RPMI medium, supplemented with 10% FBS,non-essential amino acids, sodium pyruvate, and b-mercaptoethanol withrecombinant human IL-2 at 300 IU/mL at a density of 0.25-0.3 millioncells/mL. On day 9 of expansion fresh CTS Dynabeads Treg Xpander wereadded at a 1:1 bead to cell ratio.

Transduction of Primary Tregs—Primary Tregs were transduced with CV-CARconstructs on day 2 of expansion via spin-occulation in the presence ofprotamine sulfate.

Activation of Tregs—Before start of activation cultures, CAR expressingTregs were labelled with a proliferation dye, CFSE. CV-CAR-expressingTreg cells were cultured in vitro with bead captured CV peptide. Tregactivation was assessed by measurement of percentages of proliferatingcells.

Flow Cytometry and FACS Analysis Activation Assay—Activation cultureswere collected and centrifuged at 300×g for 5 min and then resuspendedin 1× Flowstain Buffer (Invitrogen) with a viability dye (Invitrogen)and anti-EGFR surface staining antibody. Tregs were incubated for 30 minat 4° C., then centrifuged and washed with 1× Flowstain Buffer. Stainedcells were fixed with CytoFix (BD Biosciences) then analyzed by flowcytometry.

Flow Cytometry and FACS Analysis of Treg phenotype—On Day 14 ofexpansion untransduced (UTD), CD19-CAR and CV-CAR expressing Tregs werestained for transcription factors FoxP3 and Helios. Cells were fixed andpermeabilized using eBiosciences FoxP3 transcription factor buffer set(eBiosciences).

Resting of Treg Cultures—On day 14 of expansion CD19-CAR and CV-CARexpressing Tregs were harvested, de-beaded using a magnet and put inculture with 300 IU/ml IL-2 only at 0.5 million cells/ml

Flow Cytometry and FACS Analysis of CAR Expression—CD19-CAR and CV-CARexpressing Tregs on day 14 of expansion and after 2 or 5 days of restwere stained with viability dye (Invitrogen) and anti-EGFR surfacestaining antibody. CV-CAR expression was detected by incubating thecells with 1 μg/ml CV, followed by FITC-labelled anti-vimentin (cloneV9, Invitrogen).

Plate Coating—Citrullinated vimentin (CV) was diluted in PBS and addedto wells of a black/white isoplate. Plates were placed at 4° C.overnight to coat the plate wells.

Luciferase Assay—Stably-transduced and untransduced (UTD) Jurkat-FF-luccell lines were pelleted and resuspended in RPMI containing 10% FBS. Thecitrullinated protein-coated plates were washed three times with PBS.50,000 cells per well in 75 μl were added the coated plates. 5 μl of15×PMA/Iono in RPMI were added to each well, and the plates were placedin the 37° C. incubator. Approximately 24 hours later, 75 μl BioGloreagent was added to each well, and the plates were incubated 2-3 minsin the dark. Luminescence was then read on a plate reader.

FIG. 10A shows that both EF1A and MND promoters drive expression andfunctional responses by CV-CAR T cells to soluble bead-boundcitrullinated peptide (pCV). FIG. 10B shows that the use of differentpromoters does not change the phenotype of CV-CAR Treg cells.

FIG. 11 shows that the choice of scFv linker has little to no impact onSBT01G CAR T cell responses to full length CV.

FIG. 12A shows that transduction with the SBT01G CAR vector resulted inCV-CAR expression in a higher percentage of cells than did transductionwith the BVCA1 CAR vector. FIG. 12B shows that SBT01G CAR and BVCA1 CARTreg cells have similar FoxP3 and Helios profiles.

Thus, FIGS. 10-12 show that SBT01G showed higher percentage ofCAR-positive Tregs, and SBT01G showed higher levels of CAR expression.

Example 5: Assessment of CV-CAR Treg Activation by CitrullinatedVimentin In Vitro

Treg Isolation and Tissue Culture—Primary human Treg cells were sourcedfrom healthy donors from leukoreduction chamber residuals or leukopaks.Peripheral blood mononuclear cells (PBMC) were isolated by densitygradient centrifugation using Ficoll-Paque Plus. CD25+ cells wereenriched by positive selection. Treg cells were next isolated using FACSby gating for CD4+CD25+CD127lo cells. After isolation, cells werestimulated with CTS Dynabeads Treg Xpander (Gibco) at a 1:1 bead to cellratio and cultured in RPMI medium, supplemented with 10% FBS,non-essential amino acids, sodium pyruvate, and penicillin/streptomycinwith recombinant human IL-2 at 300 IU/mL at a density of 0.25-0.3million cells/mL.

Activation of Tregs—Untransduced and CV-CAR-expressing Treg cells werecultured in vitro with the citrullinated vimentin antigen ranging indose from 10 ng/mL to 10 μg/mL. Treg activation was assessed bymeasurement of percentages of proliferating cells, and levels of CD71expression and IL-10 secretion.

Flow cytometry and FACS analysis: Untransduced and CV-CAR-expressingTreg cells were collected and centrifuged at 300×g for 5 min and thenresuspended in lx RoboSep Buffer (StemCell Technologies) with a CD71surface staining antibody and Cell Trace Violet (CTV) viability dyecocktail (Invitrogen). Tregs were incubated for 30 min at 4° C., thencentrifuged and washed with 1× RoboSep Buffer. Stained cells were thenanalyzed by flow cytometry.

Results

Treg activation in response to citrullinated vimentin (CV) was assessedin vitro. FIG. 13 shows a dose-dependent, CV-induced activation ofCV-CAR Treg cells, as demonstrated by increases in proliferating cells,CD71 expression and IL-10 secretion. In contrast, untransduced Tregcells were not activated by CV. These results demonstrate that Tregactivation upon stimulation with CV is specific to Tregs expressing aCV-CAR.

The amount of CV present in the synovial fluid of healthy donors andrheumatoid arthritis (RA) patients was assessed by ELISA as shown inFIG. 14 . Jurkat reporter cells expressing one of three differentCV-CARs or a control CD19-CAR were incubated with increasing amounts ofsynovial fluid and responses were measured as an indicator of CV-CARbinding and signal transduction for T cell activation. FIG. 14 showsthat Jurkat cells (immortalized, human T lymphocytes) expressing CV-CAR1(BVCA1) and CV-CAR2 (SBT01G), but not CV-CAR3 (C03) or CD19-CAR, showedan increase in response to synovial fluid (SF). FIG. 14 also shows thatCV-CAR2 (SBT01G) T cells were activated by SF samples from more RApatients than CV-CAR1 (BVCA1) T cells.

In order to demonstrate that Tregs expressing CV-CARs are activated bycitrullinated antigen in the synovial fluid of RA patients, untransducedand CV-CAR2 Tregs were incubated with RA synovial fluid or vehicle andlevels of proliferation and CD71 expression were measured. FIG. 15 showsthat CV-CAR2 Treg cells from two donors were activated in a RA synovialfluid-specific manner.

Taken together, these results establish that CV-CAR Treg cells are ableto bind to a citrullinated antigen, which is present in RA synovialfluid, resulting in activation necessary for suppressive functions.

Example 6: Assessment of CV-Induced Suppression of Teff Cells by CV-CARTregs

Treg suppression assay—Treg cells expressing CV-CARs were prepared asdescribed above from fresh human leukopaks in a 96-well U-bottom plate.Tregs were incubated with CV as antigenic stimulus or with a vehiclecontrol. Tregs were co-cultured with either CD3/CD28-activated orCD19-CAR-specific Teff cells to assess Treg suppression of target cells.Teff cells were co-cultured at the following Treg:Teff ratios:(CD3/CD28-activation) 1:4=0.25, 1:2=0.5, 1:1=1, 2:1=2 and 4:1=4, or(CD19-specific) 1:4, 1:2, 1:1, 2:1 and 4:1.

Results

FIG. 16A shows that CV-CAR Tregs stimulated with CV were able tosuppress proliferation of CD3/CD28 activated Teff cells at a lowTreg:Teff ratio. FIG. 16B shows that CV-CAR Tregs stimulated with CVwere also able to suppress proliferation of CD19-CAR Teff cells at a lowTreg:Teff ratio.

Example 7: Analysis of CV-CAR Tregs in a Rodent Model of CV-AssociatedPulmonary Inflammation

In human rheumatoid arthritis (RA) patients, consistently highconcentrations of citrullinated vimentin (CV) in synovial fluid identifyCV as a relevant biomarker for human RA. However, current mouse modelsof RA display inconsistent CV levels, which complicates the in vivoanalysis of CV-CAR Tregs. To induce CV production and release into theextracellular matrix, a mouse model of LPS-induced lung inflammation wasdeveloped. The model is characterized by an acute inflammatory responseincluding pulmonary neutrophilia and increased secretion of thepro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1beta(IL-1b) and tumor-necrosis factor-alpha (TNF-alpha), as well as anaccumulation of CV in lung tissue within days after intranasal LPSchallenge. Chimeric antigen receptors (CAR) specific to CV have beenshown to react to the accumulated CV protein in lung tissue, whichresults in survival, proliferation and increases in suppressive activityin an antigen-specific manner of the respective CV-CAR Treg cells.

Methods

Six-to-eight week old immunocompromised NCG mice were obtained fromCharles River Laboratories. Mice were randomized by body weight anddivided into groups of 5-10 animals. Mice were subjected to intranasal(IN) treatment with LPS (5 mg/kg) on days 0, 1, 6, and 12 to inducepulmonary inflammation, and release and accumulation of citrullinatedvimentin (CV) in the affected tissues. On day 0, CV-CAR Treg cells wereharvested from actively growing cultures, the stimulation beads wereremoved magnetically, and the Tregs were subsequently labeled with CellTrace Violet (CTV) (Invitrogen). About 4 hours after the initial LPSchallenge, the CTV-labeled Tregs were adjusted to 25×10⁶ cells/mL in0.9% sterile saline solution (NaCl) and a 200 μl dose was injectedintravenously (IV) such that each mouse received a total of 5×10⁶ cells.Following CV-CAR Treg or control CD19-CAR Treg adoptive cell transfer,all mice received intraperitoneal (IP) injections of IL-2 (160,000 IU)on days 1, 2, 5, 6, 7, 8, 9, and 12. Mice were monitored daily for anyclinical signs of distress, and body weight was measured twice per week.Mice were euthanized on day 13, and the spleen and lungs were harvestedand processed into single cell suspensions for flow cytometric analysisof the human CV-CAR and CD19-CAR Tregs.

Results

FIG. 17 shows an exemplary timeline for inducing CV-associated,pulmonary inflammation in mice and the activation of CV-CAR Treg cellsin vivo.

Results

FIG. 18A shows how a proliferation ratio was calculated from flowcytometry data, while FIG. 18B show how a fold change in EGFR ratio (CARmarker) was calculated from the same data.

FIG. 19A shows that the absolute number of CV-CAR Tregs weresignificantly enriched in lung tissue from mice with LPS-inducedpulmonary inflammation. FIG. 19B shows that the proliferation ratio ofCV-CAR Tregs was significantly increased in lung tissue from mice withLPS-induced pulmonary inflammation. These results demonstrate thatCV-CAR Tregs have improved capacity compared to control Tregs to hone toand proliferate in inflamed lung tissues characterized by accumulationof citrullinated vimentin. As such, this murine pulmonary inflammationmodel permits the testing of human CV-CAR Tregs in vivo.

EXEMPLARY EMBODIMENTS

-   -   1. A chimeric antigen receptor (CAR) comprising an        antigen-binding domain, a hinge domain, a transmembrane domain,        one or more co-stimulatory domains, and an intracellular        signaling domain, wherein the antigen binding domain        specifically binds to a citrullinated polypeptide, optionally        wherein the antigen-binding domain specifically binds to three        or more different citrullinated proteins or citrullinated        fragments thereof.    -   2. The CAR of Embodiment 1, wherein the antigen binding domain        binds to a plurality of different citrullinated proteins or        citrullinated fragments thereof.    -   3. The CAR of Embodiment 1, wherein the antigen binding domain        binds to one or more of: (i) citrullinated vimentin, (ii)        citrullinated filaggrin, (iii) citrullinated fibrinogen and (iv)        citrullinated peptides fragments of these, e.g., of at least any        of 10, 12 , 14 or 16 amino acids.    -   4. The CAR of Embodiment 1, wherein the antigen-binding domain        binds to at least two of (i) citrullinated vimentin, (ii)        citrullinated filaggrin, and (iii) citrullinated fibrinogen, or        citrullinated peptides fragments thereof.    -   5. The CAR of Embodiment 1, wherein the antigen-binding domain        binds to all three of (i) citrullinated vimentin, (ii)        citrullinated filaggrin, and (iii) citrullinated fibrinogen, or        citrullinated peptides fragments thereof.    -   6. The CAR of Embodiment 5, that binds further binds        citrullinated tenascin C.    -   7. The CAR of Embodiment 1, wherein the antigen binding domain        comprises a VH domain and a VL domain, wherein:        (i) the VH domain comprises a VH-CDR1 comprising the amino acid        sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid        sequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino        acid sequence of SEQ ID NO:36, and        (ii) the VL domain of the target-binding domain comprises a        VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a        VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and        a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:43.    -   8. The CAR of Embodiment 1, wherein the intracellular signaling        domain is derived from CD3zeta.    -   9. The CAR of Embodiment 1, wherein the at least one        co-stimulatory domain is derived from a member of the group        consisting of: FceR1g, Fcg, CD28, 4-1BB, CTLA-4, CTLA-4/CD-28        hybrid, DAP10, CD27, and 2B4, optionally wherein the at least        one co-stimulatory domain comprises a CD28 and/or a 4-1BB        co-stimulatory domain.    -   10. The CAR of Embodiment 1, wherein the antigen binding domain        comprises an antibody, antibody fragment, a camelid nanobody, a        heavy chain only antibody or an aptamer.    -   11. The CAR of any one of Embodiments 1 to 10, wherein the        transmembrane domain is a CD8 transmembrane domain or a CD28        transmembrane; and the hinge domain is a CD8 hinge domain or a        CD28 hinge domain; and/or further comprising a signal peptide,        optionally wherein the signal peptide is a CD8 signal peptide or        a GM-CSF signal peptide.    -   12. The CAR of Embodiment 11, wherein the antigen-specific        binding domain comprises a single chain fragment.    -   13. The CAR of Embodiment 12, wherein the single chain fragment        comprises a single chain variable fragment (scFv), optionally        wherein the scFv fragment comprises:        (a) a VH domain comprising the amino acid sequence of SEQ ID        NO:1; and        (b) a VL domain comprising the amino acid sequence of SEQ ID        NO:4.    -   14. The CAR of Embodiment 13, wherein the scFv fragment        comprises:

(a) a VH selected from: (1) SBT01 VH (M) (SEQ ID NO: 1)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA RLDPFDYWGRGTLVTVSS; or(2) SBT01 VH (G) (SEQ ID NO: 2)QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA RLDPFDYWGRGTLVTVSS;and (b) a VL selected from: (1) SBT01 VL (M) (SEQ ID NO: 3)SYVLTQPPSVSLAPGETATITCGGDDIENQNVNWYQQKSGQAPMLLIFFDTRRPSGIPERFSGSRSEDTANLTITRVEAGDDADYFCQVYDRKTDHQV FGPGTTVTVL or(2) SBT01 VL (G) (SEQ ID NO: 4)SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQV FGTGTKVTVL.

-   -   15. The CAR of Embodiment 14, wherein the VH-VL fragments are        joined by a linker selected from the group consisting of (i)        GGGSx3 (SEQ ID NO:20), (ii) Whitlow 218 (SEQ ID NO:21),        and (iii) AB Pur (SEQ ID NO:22).    -   16. The CAR of Embodiment 13, wherein the scFv comprises the        amino acid sequence of:

(a) SBT01G-VHVL-GGGSx3 Linker-pSB_0149 (SEQ ID NO: 5)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA RLDPFDYWGRGTLVTVSSGGGGSGGGGSGGGGS SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (GGGSx3 linker underlined);or (b) SBT01G-VHVL-Whitlow 218 Linker-pSB_0158 (SEQ ID NO: 6)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA RLDPFDYWGRGTLVTVSSGSTSGSGKPGSGEGSTKG SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR(Whitlow 218 Linker underlined); or(c) SBT01G-VHVL-AB pur Linker-pSB_0159 (SEQ ID NO: 7)HLHLQESGPGLVKPSETLSLTCTVSGGSINDTTYYWGWIRQPPGKGLEWIGSIYYRGNTHYNSSLRSRVTMSVDTSKNRFSLKVTSVTAADTAVYYCA RLDPFDYWGRGTLVTVSSASSGGSTSGSGKPGSGEGSSGSAR SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHQVFGTGTKVTVLR (AB pur Linker underlined).

-   -   17. The CAR of any of Embodiments 1-16, wherein the        co-stimulatory domain comprises a CD28, 41BB, OX40, OX40, CD40L,        MyD88, CD40, CD27, ICOS, or RANK/TRANCE-R co-stimulatory domain.    -   18. A nucleic acid encoding the CAR of any of Embodiments 1-17.    -   19. The nucleic acid of Embodiment 18, comprising DNA or RNA.    -   20. The nucleic acid of Embodiment 18, wherein:        the VH is encoded by the nucleic acid sequence comprising:

CACCTGCACTTGCAGGAGTCGGGCCCAGGACTTGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAACGATACCACTTACTACTGGGGCTGGATTCGCCAGCCCCCCGGGAAGGGACTGGAGTGGATTGGGAGTATCTATTACCGGGGGAACACCCACTACAATTCGTCCCTGAGGAGTCGCGTCACCATGTCTGTCGACACTTCCAAGAACCGATTCTCCCTGAAGGTCACTTCTGTGACTGCCGCAGACACGGCTGTCTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGC, orCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACTCGACCCATTTGACTACTGGGGCCGTGGCACCCTGGTCACTGTCT CGAGC;andthe VL is encoded by a nucleic acid sequence comprising;

TCCTATGTCCTGACTCAGCCACCCTCAGTGTCGCTGGCCCCGGGAGAGACGGCCACAATTACTTGTGGTGGAGACGACATTGAAAATCAAAATGTCAACTGGTATCAGCAGAAGTCAGGTCAGGCCCCTATGCTGCTCATCTTCTTTGATACCAGACGGCCCTCAGGGATCCCGGAGCGATTCTCTGGCTCCAGGTCTGAGGACACGGCCAACCTGACCATCACCAGGGTCGAGGCCGGGGATGACGCCGACTATTTCTGTCAGGTGTATGATAGGAAGACTGATCACCAAGTCTTCGGACCTGGGACCACGGTCACCGTCCTA, orTCCTATGTGCTGACTCAGCCACCCTCAGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTCATCTATTATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGACAGTAGTAGTGATCACCAAGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA.

-   -   21. The nucleic acid of Embodiment 20, wherein the VH and VL        fragments are joined by linker and comprise the nucleic acid        sequence of (i) SEQ ID NO:12, (ii) SEQ ID NO:13, or (iii) SEQ ID        NO:14.    -   22. An expression vector comprising an expression control        sequence operatively linked to the nucleic acid sequence of any        of Embodiments 16-21.    -   23. The expression vector of Embodiment 22, wherein the        expression control sequence comprises a regulatory region,        wherein the regulatory region is selected from the group        consisting of: promoter sequences, enhancer sequences, response        elements, protein recognition sites, inducible elements, protein        binding sequences, 5′ and 3′ non-translated regions,        transcriptional start sites, termination sequences,        polyadenylation sequences, nuclear localization, signals, and        introns.    -   24. The expression vector of Embodiment 23, wherein the vector        is an adenoviral vector, a lentiviral vector or a plasmid.    -   25. A host cell comprising the expression vector of Embodiment        22, 23 or 24.    -   26. A modified T cell that has been modified to express the        chimeric antigen receptor (CAR) of any of Embodiments 1-17.    -   27. The modified T cell of Embodiment 26, wherein the T cell is        a mammalian T cell.    -   28. The modified T cell of Embodiment 27, wherein the T cell is        a Treg cell.    -   29. The modified T cell of Embodiment 28, wherein the T-cell is        a human T cell.    -   30. The modified T cell of Embodiment 29, wherein the T cell is        a primary T cell.    -   31. The modified T cell of Embodiment 30, wherein the T cell is        CD4+, CD25+ and CD127lo.    -   32. A pharmaceutical composition comprising a plurality of the        modified T cell of any of Embodiments 26 to 31, and a        pharmaceutically acceptable carrier.    -   33. A method of treating a subject suffering from rheumatoid        arthritis, comprising administering to the subject an effective        amount of a pharmaceutical composition of Embodiment 32.    -   34. The method of Embodiment 33, wherein the subject is a human.    -   35. The method of Embodiment 33 or 34, wherein the        pharmaceutical composition is administered into a synovium of        the subject.    -   36. The method of Embodiment 33 or 34, wherein the        pharmaceutical composition is administered intravenously.    -   37A. The method of Embodiment 33, wherein administering        comprises:        (a) isolating T cells from a biological sample obtained from the        subject;        (b) enriching the T cells for T regulatory cells (Treg);        (c) transfecting the enriched Treg cells with an expression        vector comprising an expression control sequence operatively        linked to a nucleotide sequence encoding a chimeric antigen        receptor (CAR) comprising an antigen binding domain that binds        to a citrullinated polypeptide;        (d) expanding the transfected Treg cells; and (e) administering        the expanded Treg cells to the subject.    -   37B. A method of treating a subject suffering from rheumatoid        arthritis, the method comprising:        (a) isolating T cells from a biological sample obtained from the        subject;        (b) enriching the T cells for T regulatory cells (Treg);        (c) transfecting the enriched Treg cells with an expression        vector encoding the CAR of any one of Embodiments 1-17;        (d) expanding the transfected Treg cells; and        (e) administering the expanded Treg cells to the subject.    -   38. The method of Embodiment 37, wherein the expansion comprises        using anti-CD3/CD28 coated beads.    -   39. The method of Embodiment 37, wherein the expansion is does        not use anti-CD3/CD28 coasted beads.    -   40. The method of Embodiment 37, wherein the transfection occurs        by use of a viral vector, electroporation, heat shock,        bacteriophage, sonication, or calcium phosphate.    -   41. The method of any of Embodiments 37 to 40 further comprising        administering one or more anti-inflammatory and/or therapeutic        agents to the subject.    -   42. The method of Embodiment 41, wherein the anti-inflammatory        agent comprises an antibody that inhibits a pro-inflammatory        cytokine.    -   43. The method of Embodiment 42, wherein the anti-inflammatory        agent is an anti-TNF-antibody, an anti-IL-6 antibody, or a        combination thereof.    -   44. A kit comprising a container containing the pharmaceutical        composition of Embodiment 32, communicating through a fluidic        conduit with a drip chamber, wherein the drip chamber        communicates through a fluidic conduit with an intravenous        needle.    -   45. The kit of Embodiment 44, wherein the container comprises a        bag.    -   46. The kit of Embodiment 44, wherein the fluidic conduit        between the container and the needle comprises one or more        Y-sites and a roller clamp.

It should be understood that the description and the drawings are notintended to limit the invention to the particular form disclosed, but tothe contrary, the intention is to cover all modifications, equivalents,and alternatives falling within the spirit and scope of the presentinvention as defined by the appended claims. Further modifications andalternative embodiments of various aspects of the invention will beapparent to those skilled in the art in view of this description.Accordingly, this description and the drawings are to be construed asillustrative only and are for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed or omitted, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims. Headings used herein are for organizational purposesonly and are not meant to be used to limit the scope of the description.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1-43. (canceled)
 44. A kit comprising a container containing apharmaceutical composition communicating through a fluidic conduit witha drip chamber, wherein the drip chamber communicates through a fluidicconduit with an intravenous needle, and wherein the pharmaceuticalcomposition comprising a plurality of Treg cells engineered to express achimeric antigen receptor (CAR-Treg), and a pharmaceutically acceptablecarrier, the CAR comprises an antigen-binding domain, a hinge domain, atransmembrane domain, one or more co-stimulatory domains, and anintracellular signaling domain, the antigen binding domain binds tocitrullinated vimentin, citrullinated filaggrin, and citrullinatedfibrinogen, or citrullinated fragments thereof, and the antigen bindingdomain comprises a VH domain and a VL domain, wherein: (i) the VH domaincomprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32,a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and aVH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and (ii) theVL domain of the target-binding domain comprises a VL-CDR1 comprisingthe amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the aminoacid sequence of SEQ ID NO:41, and a VL-CDR3 comprising the amino acidsequence of SEQ ID NO:43.
 45. The kit of claim 44, wherein the containercomprises a bag.
 46. The kit of claim 44, wherein the fluidic conduitbetween the container and the needle comprises one or more Y-sites and aroller clamp.
 47. A method of preparing T regulatory cells (Treg)expressing a chimeric antigen receptor (CAR), the method comprising: (a)isolating T cells from a biological sample obtained from a humansubject; (b) enriching the T cells for T regulatory cells (Treg); (c)transfecting the enriched Treg cells with an expression vector encodingthe CAR to produce CAR-Treg cells; and (d) expanding the CAR-Treg cellsto produce a plurality of CAR-Treg cells, wherein the CAR comprises anantigen-binding domain, a hinge domain, a transmembrane domain, one ormore co-stimulatory domains, and an intracellular signaling domain, theantigen binding domain binds to citrullinated vimentin, citrullinatedfilaggrin, and citrullinated fibrinogen, or citrullinated fragmentsthereof, and the antigen binding domain comprises a VH domain and a VLdomain, wherein: (i) the VH domain comprises a VH-CDR1 comprising theamino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acidsequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acidsequence of SEQ ID NO:36, and (ii) the VL domain of the target-bindingdomain comprises a VL-CDR1 comprising the amino acid sequence of SEQ IDNO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, anda VL-CDR3 comprising the amino acid sequence of SEQ ID NO:43.
 48. Themethod of claim 47, wherein the expansion comprises using anti-CD3/CD28coated beads.
 49. The method of claim 47, wherein the expansion does notcomprise using anti-CD3/CD28 coasted beads.
 50. The method of claim 47,wherein the transfection occurs by use of a viral vector,electroporation, heat shock, bacteriophage, sonication, or calciumphosphate.
 51. The method of claim 50, wherein the transfection occursby use of a viral vector.
 52. The method of claim 47, wherein theantigen-binding domain comprises a single chain variable fragment (scFv)comprising: (a) a VH domain comprising the amino acid sequence having atleast 95% identity to SEQ ID NO:1; and (b) a VL domain comprising theamino acid sequence having at least 95% identity to SEQ ID NO:4.
 53. Themethod of claim 52, wherein the scFv comprises the amino acid sequencehaving at least 95% identity to SEQ ID NO:5.
 54. The method of claim 47,wherein the human subject has rheumatoid arthritis.
 55. A pharmaceuticalcomposition comprising the plurality of the CAR-Treg cells produced bythe method of claim 54, and a pharmaceutically acceptable carrier.
 56. Amethod of treating rheumatoid arthritis, the method comprising:administering the to the subject an effective amount of thepharmaceutical composition of claim
 55. 57. The method of claim 56,further comprising administering one or more anti-inflammatory and/ortherapeutic agents to the subject.
 58. The method of claim 57, whereinthe anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6antibody, or a combination thereof.
 59. A method of treating rheumatoidarthritis, the method comprising administering to a subject an effectiveamount of a pharmaceutical composition, wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier and aplurality of T regulatory cells engineered to express a chimeric antigenreceptor (CAR), wherein the CAR comprises an antigen-binding domain, ahinge domain, a transmembrane domain, one or more co-stimulatorydomains, and an intracellular signaling domain, the antigen bindingdomain binds to citrullinated vimentin, citrullinated filaggrin, andcitrullinated fibrinogen, or citrullinated fragments thereof, and theantigen binding domain comprises a VH domain and a VL domain, wherein:(i) the VH domain comprises a VH-CDR1 comprising the amino acid sequenceof SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ IDNO:34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36,and (ii) the VL domain of the target-binding domain comprises a VL-CDR1comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprisingthe amino acid sequence of SEQ ID NO:41, and a VL-CDR3 comprising theamino acid sequence of SEQ ID NO:43.
 60. The method of claim 59, whereinthe antigen-binding domain comprises a single chain variable fragment(scFv) comprising: (a) a VH domain comprising the amino acid sequencehaving at least 95% identity to SEQ ID NO:1; and (b) a VL domaincomprising the amino acid sequence having at least 95% identity to SEQID NO:4.
 61. The method of claim 61, wherein the scFv comprises theamino acid sequence having at least 95% identity to SEQ ID NO:5.
 62. Themethod of claim 59, further comprising administering one or moreanti-inflammatory and/or therapeutic agents to the subject.
 63. Themethod of claim 62, wherein the anti-inflammatory agent is an anti-TNFantibody, an anti-IL-6 antibody, or a combination thereof.